CN1521180A - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins Download PDF

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CN1521180A
CN1521180A CNA2004100054154A CN200410005415A CN1521180A CN 1521180 A CN1521180 A CN 1521180A CN A2004100054154 A CNA2004100054154 A CN A2004100054154A CN 200410005415 A CN200410005415 A CN 200410005415A CN 1521180 A CN1521180 A CN 1521180A
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avec
avermectin
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deinsectization streptomycete
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��J��ʩͼ����-�����ֶ�
金·J·施图茨曼-恩格沃尔
加藤芳弘
��A��I����˰�ɪ
哈米什·A·I·麦克阿瑟
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Abstract

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product, which polynucleotide molecules can be used to alter the ratio or amount of class 2:1 avermectins produced in fermentation cultures of S. avermitilis. The present invention further relates to vectors, host cells, and mutant strains of S. avermitilis in which the aveC gene has been inactivated, or mutated so as to change the ratio or amou nt of class 2:1 avermectins produced.

Description

Mediation avermectin B2: the deinsectization streptomycete gene of B1 ratio
The present patent application is that application number is 99805027.X, and denomination of invention is " mediation avermectin B2: the deinsectization streptomycete gene of B1 ratio ", and the applying date is the dividing an application of International Application PCT/IB99/00130 on January 25th, 1999.
1. Invention field
The present invention relates to the production method of the composition and the avermectin of avermectin, be mainly used in the animal health field.More specifically, the present invention relates to include the polynucleotide molecule of the nucleotide sequence of coding aveC gene product, this polynucleotide molecule can be used to adjust avermectin 2 classes that the fermentation of deinsectization streptomycete culture is produced: the ratio of 1 class the invention still further relates to the composition and the method for screening this polynucleotide molecule.The invention further relates to carrier, transformed host cells, the novel mutant strain of deinsectization streptomycete (wherein the aveC gene by sudden change to adjust avermectin 2 classes produced: the ratio of 1 class).
2. Background of invention
2.1. avermectin
Streptomycete can produce various secondary metabolites, comprising avermectin, it contains a series of eight kinds of relevant macrocyclic lactone compounds of 16 Yuans that can produce effective anthelmintic and kill insect active, these eight kinds completely different but closely-related compounds mainly refer to: A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b." a " series compound refers to natural avermectin, and its substituting group in the C25 position is (S)-sec-butyl, and the substituting group that " b " series compound refers to the C25 position is those compounds of sec.-propyl.Code name " A " reaches " B " and refers to the avermectin that the C5 bit substituent is respectively methoxyl group and hydroxyl; Numeral " 1 " refers in the C22 position and the C23 position is the avermectin of two keys.And numeral " 2 " has a hydrogen atom reaches a hydroxyl in the C23 position avermectin for the C22 position; In these relevant avermectins, the avermectin of B1 type is acknowledged as to have the strongest parasiticide and murders the worm activity, thereby also is the avermectin of tool commercial promise.
Avermectin and the production method by deinsectization streptomycete bacterial strain aerobic fermentation thereof be at United States Patent (USP) 4,310, all has description in 519 and 4,429,042.The biosynthesizing of natural avermectin is considered to can be initial by the coenzyme A sulfo-fat analogue endogenous of isopropylformic acid and S-(+)-2-Methyl Butyric Acid.
Can effectively produce the avermectin analogue through the lipid acid that random mutagenesis carries out the bacterial strain improvement and unite the use external source providing.The mutant of the deinsectization streptomycete of side chain dioxyacetic acid desaturase defective (bkd defective mutant) only could produce avermectin when being supplemented with the lipid acid fermentation.Screening and the mutant that separates side chain dehydrogenase activity defective (as deinsectization streptomycete, ATCC 53567) existing description in European patent (EP) 276103.Exist the result of these mutant of bottom fermentation for only to produce four kinds of avermectins at the lipid acid that has external source to provide corresponding to employed lipid acid.Therefore, replenish deinsectization streptomycete (ATCC 53567) fermentation with S-(+)-2-Methyl Butyric Acid, the result produces natural avermectin A1a, A2a, B1a and B2a; Use the isopropylformic acid afterfermentation, the result produces natural avermectin A1b, A2b, B1b and B2b; And use Cyclopentane carboxylic acid afterfermentation, result to produce four kinds of novel cyclopentyl avermectin A1, A2, B1 and B2.
If can produce new avermectin with other lipid acid afterfermentation.By screening more than 800 kind of potential precursor, identified other new avermectin of kind more than 60 (example is seen Dutton etc., 1991, microbiotic magazine, 44:357-365; With Banks etc., 1994, Roy.Soc.Chem.147:16-26).In addition, in fact the deinsectization streptomycete mutant of 5-O-methyl transferase activity defective can only produce the category-B avermectin, thereby the mutant that lacks the deinsectization streptomycete of side chain dioxyacetic acid desaturase and 5-O-methyl transferase activity is simultaneously only produced the category-B avermectin corresponding to the used lipid acid of afterfermentation.Therefore, replenish this double mutant fermentation with S-(+)-2-Methyl Butyric Acid, the result only produces natural avermectin B1a and B2a, then can produce natural avermectin B1b and B2b or novel cyclopentyl B1 and B2 avermectin respectively with isopropylformic acid or Cyclopentane carboxylic acid afterfermentation.And replenish this double mutant bacterium with hexahydrobenzoic acid is the prefered method that produces commercially important new type disinsectization rhzomorph cyclohexyl avermectin B1 (doramectin).The separation of this double mutant such as deinsectization streptomycete (ATCC53692) and characteristic description are in European patent application EP 276103.
2.2. participate in the biosynthetic gene of avermectin
Under many circumstances, relate to the gene of secondary metabolite production and the position of gene on karyomit(e) of the specific antibiotic of encoding usually is bunch to gather together, for example, streptomycete polyketide synthase gene cluster (PKS) (is seen Hopwood and Sherman, 1990, heredity is commented academic year, 24:37-66).Like this, a strategy that carries out gene clone by biosynthetic pathway is to separate drug resistance gene and detect on the karyomit(e) other gene relevant with the certain antibiotics biosynthesizing in the adjacent area then.Another strategy of cloning the genes involved of important meta-bolites biosynthesizing is the mutant complementation.For example, the DNA library part of coming self energy to produce the biology of specific meta-bolites is imported into the mutant that does not produce this meta-bolites and screens the transformant that produces this meta-bolites.In addition, utilize the probe come from other streptomycete to carry out library hybridization and be used to differentiate and clone gene in the biosynthetic pathway.
Relate to the biosynthetic gene of avermectin (ave gene), (for example, PKS) the required gene of biosynthesizing is the same, and same bunch combines on the karyomit(e) to resemble other streptomycete secondary metabolite.Use carrier successfully to clone many ave genes to replenish the deinsectization streptomycete mutant that the avermectin biosynthesizing is obstructed.These genes be cloned in United States Patent (USP) 5,252, the existing description in 474.In addition, Ikeda etc., 1995, microbiotic magazine 48:532-534, described and related to C22, the chromosomal region of C23 dehydrating step (aveC) be positioned at the section of deinsectization streptomycete 4.82Kb BamHI enzyme disconnected on, and the aveC transgenation that produces the single component B2a producer has been described.Since ivermectin, a potential anthelmintic compound can be by avermectin B2a chemosynthesis, and it is more useful that the one-component producer of this avermectin B2a is considered at the commercial production ivermectin.
The minimized aveC transgenation of complexity that avermectin is produced, for example, reduce avermectin B2: the evaluation of the sudden change of B1 ratio can be simplified the production and the purifying of commercially important avermectin.
3. invention summary
The invention provides isolating polynucleotide molecule, it contains the complete aveC ORF gene of deinsectization streptomycete or its substantive part, and this isolating polynucleotide molecule lacks the next complete ORF that is positioned at downstream, aveC ORF present position on the deinsectization streptomycete karyomit(e).The isolating polynucleotide molecule of the present invention preferably includes the identical nucleotide sequence of deinsectization streptomycete aveC gene product encoding sequence with plasmid pSE 186 (ATCC 209604), perhaps same Fig. 1 (SEQ ID NO:1) aveC ORF or the identical nucleotide sequence of its substantive part.
The present invention further provides polynucleotide molecule, nucleotide sequence or its substantive portion homologous of the aveC ORF that exists among the deinsectization streptomycete aveC gene product encoding sequence homology of nucleotide sequence that it has and plasmid pSE186 (ATCC 209604) or same Fig. 1 (SEQ ID NO:1).
The present invention further provides polynucleotide molecule, aminoacid sequence or its substantive portion homologous of the aveC gene product encoding sequence amino acid sequence coded homology of nucleotide sequence coded amino acid sequence of polypeptide that it contains and plasmid pSE 186 (ATCC 209604) or same Fig. 1 (SEQ ID NO:2).
The present invention further provides isolating polynucleotide molecule, it contains the nucleotide sequence of a coding AveC homologue gene product.In preferred embodiments, isolating polynucleotide molecule contains the nucleotide sequence of coding streptomyces hygroscopicus AveC homologue gene product, and homologue gene product wherein contains just like the aminoacid sequence of SEQ ID NO:4 or its substantive part.In preferred embodiments, the isolating polynucleotide molecule of the present invention of coding streptomyces hygroscopicus AveC homologue gene product contains nucleotide sequence or its substantive part of SEQ ID NO:3.
The present invention further provides polynucleotide molecule, it contains and the streptomyces hygroscopicus nucleotide sequence homologous nucleotide sequence shown in the SEQ ID NO:3.The present invention also provides polynucleotide molecule, its nucleotide sequence coded polypeptide that contains with have a streptomyces hygroscopicus AveC homologue gene product homology of aminoacid sequence shown in the SEQ ID NO:4.
The present invention also provides oligonucleotide, it is hybridized with the polynucleotide molecule with nucleotide sequence shown in Fig. 1 (SEQ ID NO:1) or the SEQ IDNO:3, or hybridizes with the polynucleotide molecule with complementary nucleotide sequence of nucleotide sequence shown in Fig. 1 (SEQ ID NO:1) or the SEQ ID NO:3.
The present invention further provides recombinant cloning vector and expression vector, they can be used for the clone or express polynucleotide of the present invention, comprise the polynucleotide molecule of the ORF of the aveC ORF that contains deinsectization streptomycete or aveC homologue.In a nonrestrictive embodiment, plasmid pSE186 provided by the present invention (ATCC 209604) contains the ORF of deinsectization streptomycete aveC gene complete.The present invention further provides transformed host cell, it contains polynucleotide molecule of the present invention or recombinant vectors reaches new bacterial strain or the clone that obtains thus.
Purifying or isolating recombinant expressed AveC gene product or AveC homologue gene product or its substantive part and homologue thereof have basically been the present invention further provides.The present invention further provides the method that produces reorganization AveC gene product, described method comprises: helping producing under the condition of reorganization AveC gene product or AveC homologue gene product, cultivate and use the recombinant expression vector transformed host cells, and from cell culture, reclaim AveC gene product or AveC homologue gene product, the nucleotide sequence of the polynucleotide molecule that wherein said recombinant expression vector contained can encode AveC gene product or AveC homologue gene product, this polynucleotide molecule can be operated with one or more controlling elements that the control polynucleotide molecule is expressed in host cell and be linked to each other.
The present invention further provides polynucleotide molecule, its nucleotide sequence that comprises is identical with the deinsectization streptomycete AveC gene product encoding sequence of plasmid pSE186 (ATCC 209604), or it is identical with the nucleotide sequence of the deinsectization streptomycete aveC ORF shown in Fig. 1 (SEQ ID NO:1), but it further comprises one or more sudden changes wherein the polynucleotide molecule of the aveC allelotrope of wild-type inactivation and its expression contain the nucleotide sequence of sudden change the cell of deinsectization streptomycete bacterial strain (ATCC53692) for the allelic deinsectization streptomycete bacterial strain of the aveC that only expresses wild-type (ATCC 53692) cell, produce the avermectin of different ratios or amount.According to the present invention, can use this polynucleotide molecule to produce the new bacterial strain of deinsectization streptomycete, it is compared with the allelic identical bacterial strain of the aveC that only expresses wild-type, and the output of avermectin has the change that can survey.In preferred embodiments, this polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (it compares 2 classes to reduce with the allelic identical bacterial strain of the aveC that only expresses wild-type: 1 class ratio is produced avermectin).In a further preferred embodiment, this polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (it compares the avermectin that can produce the increase level with the allelic identical bacterial strain of the aveC that only expresses wild-type).In a further preferred embodiment, this polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (wherein aveC gene inactivation).
The invention provides the method that discriminating can change the deinsectization streptomycete aveC ORF sudden change of the ratio of the avermectin that is produced and/or amount.In preferred embodiments, the invention provides discriminating and can change avermectin 2 classes that produced: the method for the aveC ORF sudden change of 1 class ratio, described method comprises: (a) measure avermectin 2 classes that the deinsectization streptomycete bacterial strain cell produces: the ratio of 1 class, wild-type aveC allelotrope in the described cell is inactivation, and the polynucleotide molecule of nucleotide sequence that includes the AveC gene product of encoding mutant is imported into this cell and is expressed therein; (b) measure avermectin 2 classes of the strain cell generation identical with the deinsectization streptomycete of step (a): the ratio of 1 class, but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ 1D NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; Reach (c) avermectin 2 classes of the deinsectization streptomycete bacterial strain cell generation of comparison step (a): avermectin 2 classes that the deinsectization streptomycete bacterial strain cell of the ratio of 1 class and step (b) produces: the ratio of 1 class, if the two difference, then identify and can change avermectin 2 classes: the aveC ORF sudden change of 1 class ratio exists, in preferred embodiments, avermectin 2 classes: the ratio of 1 class reduces because of sudden change.
In another preferred embodiment, the invention provides the method that a kind of evaluation can change the aveC ORF sudden change of the avermectin amount that is produced or include the gene construct sudden change of aveC ORF, described method comprises: the amount of (a) measuring the avermectin of deinsectization streptomycete bacterial strain cell generation, wild-type aveC allelotrope in the described cell is inactivation, includes the nucleotide sequence of AveC gene product of encoding mutant or the polynucleotide molecule of gene construct that contains the nucleotide sequence of coding AveC gene product and is imported into this cell and expresses therein; (b) measure the amount of the avermectin of the strain cell generation identical with the deinsectization streptomycete of step (a), but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ 1D NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; And (c) amount of amount and the avermectin of the deinsectization streptomycete bacterial strain cell generation of step (b) of the avermectin that produces of the deinsectization streptomycete bacterial strain cell of comparison step (a), if the two difference then identifies the aveC ORF sudden change or the gene construct sudden change that can change the avermectin amount and exists.In preferred embodiments, the amount of avermectin increases because of sudden change.
The present invention further provides recombinant vectors, this carrier can be used for preparing the new bacterial strain of deinsectization streptomycete of the disinsection mychel yield with change.For example, the invention provides a kind of like this carrier, it can be used for any is included aveC gene locus on the polynucleotide molecule target deinsectization streptomycete karyomit(e) of sudden change nucleotide sequence of the present invention, perhaps inserts, and perhaps the mode with homologous recombination replaces aveCORF or its part.Yet, according to the present invention, when occurring with episomal form during site on being inserted into deinsectization streptomycete karyomit(e) except that the aveC gene or in the deinsectization streptomycete cell, the polynucleotide molecule that includes sudden change nucleotide sequence of the present invention is also being played a role aspect the biosynthesizing of regulation and control avermectin.Therefore, the present invention also provides a kind of like this carrier, its polynucleotide molecule that contains contains sudden change nucleotide sequence of the present invention, can use this carrier that polynucleotide molecule is inserted into the site except that the aveC gene on the deinsectization streptomycete karyomit(e), perhaps this carrier can episomal form occur.In preferred embodiments, gene replacement vector provided by the present invention can be used to will sudden change aveC allelotrope be inserted on the deinsectization streptomycete karyomit(e) to produce novel cell strain, it can more only express 2 classes of the allelic identical strain cell of wild-type aveC to reduce: 1 class ratio is produced avermectin.
The present invention further provides a kind of method for preparing the new bacterial strain of deinsectization streptomycete, described bacterial strain includes the allelic cell of the aveC that can express sudden change, it is compared with only expressing the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC, and the ratio and/or the amount of the avermectin that is produced change.In preferred embodiments, the present invention further provides a kind of method for preparing the new bacterial strain of deinsectization streptomycete, described bacterial strain includes the allelic cell of the aveC that can express sudden change, its with only express the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC and compare, 2 classes of the avermectin that is produced: 1 class ratio changes, described method comprises with the allelic carrier conversion of the aveC that carries sudden change deinsectization streptomycete bacterial strain cell, the gene product of the aveC allelotrope of this sudden change coding make the allelic deinsectization streptomycete bacterial strain cell of aveC of expressing sudden change with only express the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC and compare, 2 classes of the avermectin that is produced: 1 class ratio changes, select transformant, avermectin 2 classes that this transformant produces: 1 class ratio is more only expressed the allelic strain cell of wild-type aveC and is changed to some extent.In preferred embodiments, in new strain cell, avermectin 2 classes that produced: the ratio of 1 class reduces to some extent.
In a further preferred embodiment, the invention provides the method for preparing the new bacterial strain of deinsectization streptomycete, described bacterial strain comprises the cell of the avermectin that can produce the change amount, described method comprises uses the carrier that carries sudden change aveC allelotrope or contain the allelic gene construct of this aveC to transform the deinsectization streptomycete bacterial strain cell, the amount that the avermectin amount that the result of its expression is produced the deinsectization streptomycete bacterial strain cell of expression sudden change aveC allelotrope or gene construct is more only expressed the avermectin of the identical strain cell generation of the allelic deinsectization streptomycete of wild-type aveC changes to some extent, select cell transformed, the avermectin amount that the avermectin amount that this transformant produces is more only expressed the allelic strain cell generation of wild-type aveC changes to some extent.In preferred embodiments, in new strain cell, the amount of the avermectin that is produced increases to some extent.
In a further preferred embodiment, the invention provides a kind of method for preparing the new bacterial strain of deinsectization streptomycete, the aveC allelotrope that contains inactivation in these deinsectization streptomycete cells, described method comprises with making the carrier of aveC allelic inactivation transform express the allelic deinsectization streptomycete bacterial strain cell of wild-type aveC, and selects wherein the aveC allelotrope transformant of inactivation.
The present invention further provides the new bacterial strain of deinsectization streptomycete, its cell that contains is transformed by arbitrary polynucleotide molecule or carrier that contains sudden change nucleotide sequence of the present invention.In preferred embodiments, the invention provides the new bacterial strain of deinsectization streptomycete, the cell expressing that it contains the sudden change aveC allelotrope rather than the aveC allelotrope of wild-type, perhaps express the aveC allelotrope of sudden change and the aveC allelotrope of wild-type simultaneously, wherein 2 classes of the avermectin of this new strain cell generation: 1 class ratio changes for only expressing the allelic identical strain cell of wild-type aveC to some extent.In a more preferred embodiment, 2 classes of the avermectin of new strain cell generation: 1 class ratio decreases for only expressing the allelic identical strain cell of wild-type aveC.This new bacterial strain can be used for for example doramectin of avermectin that scale operation has a commercial application value.
In another preferred embodiment, the invention provides the new bacterial strain of deinsectization streptomycete, its cell that contains can be expressed the aveC allelotrope of sudden change or be contained the allelic gene construct of aveC, rather than the aveC allelotrope of wild-type, perhaps express the two simultaneously, consequently for the amount of the avermectin that produces with respect to the allelic identical strain cell of the aveC that only expresses wild-type, the amount that contains the avermectin that the allelic cell of aveC of this sudden change produces changes to some extent.In preferred embodiments, the amount of the avermectin of new cell generation increases to some extent.
In another preferred embodiment, the invention provides the new bacterial strain of deinsectization streptomycete, aveC gene inactivation in its cell that contains.The avermectin of the different spectrums that produced with wild type strain that this bacterial strain not only produces their is useful, and the complementary shaker test that whether influences disinsection mychel yield for the aveC gene of mensuration target as herein described or random mutagenesis also is very useful.
The present invention further provides the method for producing avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim avermectin, the aveC allelotrope of described cell expressing sudden change, only express the allelic identical strain cell of aveC of wild-type with not expressing sudden change aveC allelotrope and compare, the gene product of this mutation allele coding has changed 2 classes of the avermectin of the allelic deinsectization streptomycete bacterial strain cell generation of the aveC that expresses sudden change: the ratio of 1 class.In preferred embodiments, express 2 classes of the avermectin of the cell generation that suddenlys change: the ratio of 1 class reduces to some extent.The avermectin that this method provides commercially valuable is the high efficiency production method of doramectin for example.
The present invention further provides the method for producing avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim avermectin, the aveC allelotrope of described cell expressing sudden change or contain the allelic gene construct of aveC, only express the allelic identical strain cell of aveC of wild-type with not expressing sudden change aveC allelotrope or gene construct and compare, the amount of the avermectin that the aveC allelotrope of expression sudden change or the deinsectization streptomycete bacterial strain cell of gene construct produce changes to some extent.In preferred embodiments, 2 classes of the avermectin of the cell generation of expression sudden change or gene construct: the amount of 1 class reduces to some extent.
The present invention further provides the new composition of the avermectin that produces by the allelic deinsectization streptomycete bacterial strain cell of the aveC that expresses sudden change of the present invention, wherein do not express 2 classes of the avermectin that identical strain cell produced of the allelic deinsectization streptomycete of aveC of sudden change with the aveC allelotrope of only expressing wild-type: the ratio of 1 class is compared, and its 2 classes to reduce: 1 class ratio is produced avermectin.This novel avermectin composition may reside in the fermentation culture or from nutrient solution and obtains, and can part or purifying basically.
4. description of drawings
Fig. 1. contain the dna sequence dna (SEQ ID NO:1) of deinsectization streptomycete aveC ORF and the aminoacid sequence (SEQ ID NO:2) of inferring thereof.
Fig. 2. contain the plasmid vector pSE186 (ATCC209604) of the ORF of deinsectization streptomycete aveC gene complete.
Fig. 3. contain the gene replacement vector pSE180 (ATCC 209605) of the ermE gene that inserts the Sacc.Erythraea among the deinsectization streptomycete aveC ORF.
Fig. 4. with five kinds of eclipsed clays (be pSE65, pSE66, pSE67, pSE68, PSE69) clone identification comes from the BamHI restriction map of the avermectin polyketide synthase gene cluster of deinsectization streptomycete.Pointed out the relation of pSE118 and pSE119 when logical.
Fig. 5. the HPLC of the tunning of deinsectization streptomycete bacterial strain analyzes.By relatively to carry out the peak quantitative with the normal content of cyclohexyl B1.The retention time of cyclohexyl B2 is 7.4-7.7 minute, and the retention time of cyclohexyl B1 is 11.9-12.3 minute.FIG.5A. deinsectization streptomycete bacterial strain SE180-11, it has the aveC ORF of inactivation; FIG.5B. the deinsectization streptomycete bacterial strain SE180-11 that uses pSE186 (ATCC 209604) to transform; FIG.5C. the deinsectization streptomycete bacterial strain SE180-11 that transforms with pSE187; FIG.5D. the deinsectization streptomycete bacterial strain SE180-11 that transforms with pSE188.
Fig. 6. by the putative amino acid sequence (SEQ IDNO:2) of deinsectization streptomycete aveC ORF coding, ash produces the part of O RF (SEQ ID NO:5) of the aveC homologue of look streptomycete, and comes from the comparison of the aveC homologue ORF (SEQ ID NO:4) of streptomyces hygroscopicus.The Xie Ansuan residue of representing with runic is the initiation site that this protein is inferred.The conserved residues of representing with capitalization demonstrates the homology in whole three kinds of sequences, and the conserved residues of representing with lowercase demonstrates 2 kinds homology in 3 kinds of sequences.These aminoacid sequences have nearly 50% sequence identity.
Fig. 7. contain the segmental heterozygosis plasmid construction of the BsaAI/KpnI body of 564 bp that come from streptomyces hygroscopicus aveC homologue gene (it is inserted in the BsaAI/KpnI site of the aveC ORF of deinsectization streptomycete).
5. Detailed Description Of The Invention
The present invention relates to the polynucleotide molecule that identification and characterization has the nucleotide sequence of coding deinsectization streptomycete AveC gene outcome, structure can be used for screening the AveC gene outcome of sudden change to the new bacterial strain of deinsectization streptomycete of the impact of production avermectin, and finds that the AveC gene outcome of some sudden change can reduce the avermectin B2 that deinsectization streptomycete produces: the ratio of B1. As an example, the present invention will be described below has the nucleotide sequence identical with the deinsectization streptomycete AveC gene outcome coded sequence of plasmid pSE186 (ATCC 209604), perhaps have the polynucleotide molecule of nucleotide sequence of the ORF of Fig. 1 (SEQ ID NO:1), and have the polynucleotide molecule of the sudden change nucleotide sequence that gets thus. Yet the principle that the present invention illustrates can by the polynucleotide molecule that is applied to similarly other, comprise coming from other streptomycete such as the aveC homologue gene of streptomyces hygroscopicus and ash product look streptomycete etc.
5.1. the polynucleotide molecule of coding deinsectization streptomycete aveC gene outcome
The invention provides a kind of polynucleotide molecule of separation, it includes the complete aveC ORF of deinsectization streptomycete or its substantive part, and the polynucleotide molecule of this separation lacks the next complete ORF that is in downstream, aveC ORF present position in the deinsectization streptomycete chromosome.
The nucleotide sequence that the polynucleotide molecule that the present invention separates preferably includes is identical with the coded sequence of the deinsectization streptomycete AveC gene outcome of plasmid pSE186 (ATCC 209604), and perhaps nucleotide sequence or its substantive part with the ORF of Fig. 1 (SEQ ID NO:1) is identical. " the substantive part " of the polynucleotide molecule of the separation of the nucleotide sequence that contains coding deinsectization streptomycete AveC gene outcome used herein refers to the polynucleotide molecule of separation, it comprises the about 70% of the complete aveC ORF sequence shown in Fig. 1 (SEQ ID NO:1) at least, and the AveC gene outcome that is equal to of encoding function. Here, the AveC gene outcome of " functional equivalent " is defined as gene outcome, when it was expressed among deinsectization streptomycete bacterial strain ATCC 53692 of natural aveC allelic inactivation therein, the avermectin that the resultant avermectin wild type functional aveC allelic deinsectization streptomycete bacterial strain ATCC 53692 natural with only expressing deinsectization streptomycete bacterial strain ATCC 53692 produces had substantially the same ratio and amount.
Except the nucleotide sequence of aveC ORF, the polynucleotide molecule that the present invention separates may further include natural flank in the nucleotide sequence of deinsectization streptomycete original position aveC gene, for example the flanking nucleotide sequence shown in Fig. 1 (SEQ ID NO:1).
Term used herein " polynucleotide molecule ", " polynucleotide sequence ", " coded sequence ", " ORFs " reaches " ORF ' " all means out dna molecular and RNA molecule, it can be strand or two strands, it can be transcribed or be translated (DNA), perhaps be translated (RNA) and become the AveC gene outcome, perhaps, as described below, be translated into AveC homologue gene outcome, or be translated into polypeptide (when under the control that places the appropriate regulation element, itself and AveC gene outcome or AveC homologue gene outcome homology in suitable host cell expression system). Coded sequence includes but not limited to the protokaryon sequence, cDNA sequence, genomic dna sequence, and DNA and the RNA sequence of chemical synthesis.
Nucleotides sequence shown in Fig. 1 (SEQ ID NO:1) is listed in bp position 42,174,177 and 180 and comprises four different GTG codons. SEQ ID NO:1) 5 ' a plurality of disappearances in zone can be used as the initiation site of protein expression to help to determine in these codons which in aveC ORF shown in following the 9th part, can make up aveC ORF (Fig. 1:. The disappearance in first GTG site, bp 42 positions can not be eliminated the activity of AveC. In addition, all GTG codons that lack again bp position 174,177 and 180 could be eliminated the activity of AveC, and this shows that this zone is necessary for protein expression. Therefore the present invention has comprised aveC ORF and the corresponding polypeptide thereof of different length, and described aveC ORF can start translation in arbitrary GTG site of the bp 174,177 shown in Fig. 1 (SEQ ID NO:1) or 180 bp.
The present invention further provides polynucleotide molecule, its nucleotide sequence that comprises is with the deinsectization streptomycete AveC gene outcome coded sequence homology of plasmid pSE186 (ATCC 209604), or with deinsectization streptomycete aveC ORF nucleotide sequence or its substantive homeologous shown in Fig. 1 (SEQ ID NO:1). Term " homology " is when the polynucleotide molecule that is used in reference to deinsectization streptomycete AveC gene outcome coded sequence homology, mean the nucleotide sequence that this polynucleotide molecule has: (a) the coding AveC gene outcome identical with the deinsectization streptomycete AveC gene outcome coded sequence of plasmid pSE186 (ATCC 209604), or the coding identical AveC gene outcome coded with the aveC ORF nucleotide sequence of the deinsectization streptomycete shown in Fig. 1 (SEQ ID NO:1), but its degeneracy according to genetic code has been done one or more reticent changes to nucleotide sequence; Perhaps (b) is under the tight condition of moderate, complementary sequence hybridization with following polynucleotide molecule, what this polynucleotide molecule contained is nucleotide sequence coded by the coded amino acid sequence of plasmid pSE186 (ATCC 209604) AveC gene outcome coded sequence, the AveC gene outcome of the perhaps amino acid sequence shown in the code pattern 1 (SEQ ID NO:2), and coding functional equivalent as hereinbefore defined. The tight condition of described moderate also namely under 65 ℃, 0.5M NaHPO4,7% lauryl sodium sulfate (SDS), hybridize with the DNA that is incorporated on the filter membrane among the 1mM EDTA, and under 42 ℃, washing in 0.2 * SSC/0.1%SDS (example is seen (volumes) such as Ausubel, 1989, the molecular biology fresh approach, Vol.1, Green Publishing Associates company, and John Wiley ﹠ Sons company, New York, p.2.10.3). In preferred embodiments, the polynucleotide molecule of homology under highly tight condition with plasmid pSE186 (ATCC 209604) in the complementary sequence hybridization of nucleotide sequence of coding AveC gene outcome, perhaps with the complementary sequence hybridization of the substantive part of the nucleotide sequence shown in Fig. 1 (SEQ ID NO:1) or its, and the AveC gene outcome of coding functional equivalent as hereinbefore defined. Highly tight condition refers at 65 ℃, 0.5M NaHPO4, 7% lauryl sodium sulfate (SDS) with the DNA hybridization that is incorporated on the filter membrane, reaches under 68 ℃ among the 1mM EDTA, washing in 0.1 * SSC/0.1%SDS (Ausubel etc., 1989, see above).
As described in embodiment hereinafter, the activity of AveC gene outcome and potential function equivalent thereof can be measured by the mode of analyzing tunning with HPLC. No matter be natural or artificial synthetic, polynucleotide molecule (function equivalent of the nucleotide sequence coded deinsectization streptomycete AveC gene outcome that it contains) comprises the natural AveC gene that is present in other bacterial strain of deinsectization streptomycete, is present in the aveC homologue gene of other kind of streptomycete and the aveC allele of sudden change.
The present invention further provides polynucleotide molecule, the amino acid sequence of the AveC gene outcome coded sequence of the amino acid sequence that the coded polypeptide of its nucleotide sequence that comprises contains and plasmid pSE186 (ATCC 209604) coding, or with the amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or its substantive part homology is arranged. " the substantive part " of the amino acid sequence of Fig. 1 used herein (SEQ ID NO:2) refers to this polypeptide and comprises at least about 70% the amino acid sequence shown in Fig. 1 (SEQ ID NO:2), and formed the as defined above AveC gene outcome of functional equivalent.
The amino acid sequence that this paper is used in reference to the AveC gene outcome that comes from deinsectization streptomycete has the term " homology " of the amino acid sequence of homology to refer to the polypeptide of the AveC gene outcome coded sequence coding of plasmid pSE186 (ATCC 209604), or contain polypeptide just like the amino acid sequence shown in Fig. 1 (SEQ ID NO:2) (but wherein one or more amino acid residues replaced by different amino acid residues is conservative, this conservative result who replaces has produced the gene outcome of aforesaid functional equivalent). Conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is well-known in the art. Carry out the rule description of such replacement in Dayhof, M.D., 1978, Nat.Biomed.Res.Found., Washington, D.C., Vol.5, Sup.3 etc. More specifically, conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor generally occurs in and acidity, and polarity is in the amino acid family that its side chain size is correlated with. The amino acid of gene code generally is divided into four groups: (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; And (4) uncharged polarity=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine also may be generically referred to as the amino acid of fragrance. One or more replacements in any particular group, for example, replace leucine with isoleucine or valine, perhaps replace aspartic acid with glutamic acid, perhaps replace threonine with serine, or replace any other amino acid residue with the amino acid residue of structurally associated. For example, have similar acidity, polarity, the amino acid residue of the similitude of side chain size or its some combination replaces for the not significant impact of the function of whole polypeptide.
The present invention further provides the polynucleotide molecule that separates, the nucleotide sequence coded aveC homologue gene outcome that it comprises. " AveC homologue gene outcome " used herein is defined as a gene outcome, its with contain deinsectization streptomycete AveC gene outcome by the amino acid sequence of the AveC gene outcome coded sequence of plasmid pSE186 (ATCC 209604) coding and have at least 50% amino acid sequence identity, perhaps have at least 50% amino acid sequence identity with the amino acid sequence shown in Fig. 1 (SEQ ID NO:2). In a nonrestrictive embodiment, AveC homologue gene outcome comes from streptomyces hygroscopicus (european patent application 0298423 existing the description; Preserving number is FERM BP-1901), it includes amino acid sequence or its substantive part of SEQ ID NO:4. " the substantive part " of the amino acid sequence of SEQ ID NO:4 refers to the polypeptide that contains at least 70% SEQ ID NO:4 amino acid sequence, and it consists of the AveC homologue gene outcome of functional equivalent. " functional equivalent " AveC homologue gene outcome is defined by gene outcome, when in streptomyces hygroscopicus bacterial strain FERM BP-1901, expressing (wherein natural AveC homologue allele inactivation), cause the ratio of the milbemycin that produces and amount basically to be same as and only express ratio and the amount that the streptomyces hygroscopicus bacterial strain FERM BP-1901 natural allelic streptomyces hygroscopicus bacterial strain of the functional aveC homologue of wild type FERM BP-1901 produce. In a non-limiting embodiments, the polynucleotide molecule that the present invention separates (its coding streptomyces hygroscopicus AveC homologue gene outcome) contains nucleotide sequence or its substantive part of SEQ ID NO:3. In this, the polynucleotide molecule that " the substantive part " of polynucleotide molecule of separation that contains the nucleotide sequence of SEQ ID NO:3 refers to this separation comprises 70% the nucleotide sequence shown in SFQ ID NO:3 at least, and the AveC homologue gene outcome that function is suitable as defined above of encoding.
The present invention further provides polynucleotide molecule, its nucleotide sequence that comprises is with the streptomyces hygroscopicus nucleotide sequence homology shown in the SEQ ID NO:3. Term " homology " is when being used in reference to the polynucleotide molecule that contains with the nucleotide sequence of the streptomyces hygroscopicus AveC homologue gene outcome coded sequence homology of SEQ ID NO:3, mean the nucleotide sequence that this polynucleotide molecule has: (a) the coding gene outcome identical with the nucleotide sequence of SEQ ID NO:3 or its substantive part, but its degeneracy according to genetic code has been done one or more reticent variations to nucleotide sequence; Perhaps (b) is under the tight condition of moderate, with the complementary sequence hybridization of the polynucleotide molecule of the nucleotide sequence of the amino acid sequence with coding SEQ ID NO:4 and the AveC homologue gene outcome that the above-mentioned functions of encoding is equal to, the tight condition of described moderate also namely under 65 ℃, 0.5M NaHPO4, 7% lauryl sodium sulfate (SDS) with the DNA hybridization that is incorporated on the filter membrane, reaches under 42 ℃ washing (example is seen Ausubel etc., and document is the same) among 0.2 * SSC/0.1%SDS among the 1mM EDTA. In preferred embodiments, the polynucleotide molecule of homology under highly tight condition with SEQ ID NO:3 in the nucleotide sequence of coding AveC homologue gene outcome or the complementary sequence hybridization of its substantive part, and the AveC homologue gene outcome that is equal to of coding above-mentioned functions, described height stringent condition refers at 65 ℃, 0.5M NaHPO4, 7% lauryl sodium sulfate (SDS) with the DNA hybridization that is incorporated on the filter membrane, reaches under 68 ℃ among the 1mM EDTA, washing among 0.1 * SSC/0.1%SDS (Ausubel etc., 1989, see above).
The present invention further provides a polynucleotide molecule, the polypeptide that its nucleotide sequence that comprises is coded and streptomyces hygroscopicus AveC homologue gene outcome have homology. The term " homology " that this paper is used in reference to from the AveC homologue gene outcome of the SEQ ID NO:4 that comes from streptomyces hygroscopicus has a polypeptide of homology refers to the polypeptide that contains just like amino acid sequence shown in the SEQ ID NO:4 (but wherein one or more amino acid residues replaced by different amino acid residues is conservative, this conservative result who replaces has been to produce the homologue gene outcome of aforesaid functional equivalent).
The present invention further provides oligonucleotide molecules, itself and the polynucleotide molecule hybridization that has such as the nucleotide sequence of Fig. 1 (SEQ ID NO:1) or SEQ ID NO:3 are perhaps with the polynucleotide molecule hybridization that has such as the kernel of complementary sequence nucleotide sequence of the nucleotide sequence of Fig. 1 (SEQ ID NO:1) or SEQ ID NO:3. The length of this oligonucleotides is at least about 10 nucleotides, and preferably be about 15-30 nucleotides, it is hybridized with aforementioned a kind of polynucleotide molecule under highly tight condition, namely about 37 ℃, oligonucleotides in 6 * SSC/0.5% sodium pyrophosphate about 14 bases of washing, at the oligonucleotides about 17 bases of washing about 48 ℃, at the oligonucleotides about 20 bases of washing about 55 ℃, and at the oligonucleotides about 23 bases of washing about 60 ℃. In a preferred embodiment, the part of one of this oligonucleotides and aforementioned polynucleotide molecule is complementary. These oligonucleotide molecules can be used for different purposes, comprise coding, or are used as the used antisense nucleic acid molecule of gene regulation, or are used as the primer of the polynucleotide molecule of amplification coding aveC or aveC homologue.
In addition, can be in conjunction with known technology, use polynucleotide molecule disclosed herein or oligonucleotides in other kind of streptomycete or bacterial strain, to identify other aveC homologue gene. For example, contain Fig. 1 (SEQ ID NO:1) but the part of deinsectization streptomycete nucleotide sequence or the oligonucleotide molecules of the part of the streptomyces hygroscopicus nucleotide sequence of SEQ ID NO:3 can and be used for the constructed genomic library of DNA that the screening origin comes from related organisms by the geodetic mark). The selection of hybridization conditions stringency is based on the correlation of related organism, in this example, refers to the relation of deinsectization streptomycete or streptomyces hygroscopicus and other related organisms. Requirement to different stringent conditions is that those skilled in the art are well-known, and such condition will obtain according to deriving the difference of the specific organism of library and flag sequence and carry out predictable change. The length of preferred described oligonucleotides is at least about 15 nucleotides, and it comprises, those that describe such as following embodiment. Can utilize these or other oligonucleotides to carry out homologue gene magnification by standard techniques such as using polymerase chain reactions (PCR), but also can use other amplification technique known in the art, such as ligase chain reaction.
Can detect the ability that is accredited as the clones coding function AveC homologue gene outcome that includes aveC homologue nucleotide sequence. For this reason, can carry out sequence analysis to differentiate suitable reading frame and initial sum termination signal to this clone. Perhaps or in addition, clone's dna sequence dna can be inserted suitable expression vector, namely contain the carrier of the necessary element of protein coding sequence of transcribing and translate insertion. Can be by in the multiple host/vector system of following use any, including, but not limited to such as plasmid, bacteriophage, or the bacterial system of clay expression vector etc. Can be as described in following the 7th part, by with as HPLC analyze the AveC type activity that tunning is analyzed the suitable host cell that transforms with the carrier that contains potential aveC homologue coded sequence.
The production of polynucleotide molecule as herein described and operation are that those skilled in the art are easy to accomplish, it can operate according to following recombinant technique, for example, Maniatis etc., 1989,Molecule The cloning experimentation guide, publishing house of cold spring harbor laboratory, cold spring port, New York; Ausubel etc., 1989,The modern molecular biology study course, Greene Publishing Associates ﹠ Wiley Interscience, New York; Sambrook etc., 1989,The molecular cloning experiment guide, (second edition), publishing house of cold spring harbor laboratory, cold spring port, New York; Innis etc. (volume), 1995,PCR Strategy, Academic Press company, San Diego; And Erlich (volume), 1992,Round pcr, Oxford University Press, New York (listing this paper in as a reference). The polynucleotides clone of coding AveC gene outcome or AveC homologue gene outcome can differentiate by any method known in the art, includes but not limited to the cited method of following the 7th part. By using such as Benton and Davis, 1977, the method of the screening phage library described in the science 196:180 and Grunstein and Hogness, 1975, Proc.Natl.Acad.Sci.USA, the method of the described screening plasmid library of 72:3961-3965 can be screened aveC and aveC homologue coded sequence from genome dna library. Be present in plasmid pSE186 (ATCC 209604), or among the plasmid pSE119, and have known packets contain the polynucleotide molecule (the 7th part is described below) of the nucleotide sequence of aveC ORF can be as the probe of these screening experiments. Perhaps, can synthesize correspondence by the oligonucleotide probe of the nucleotide sequence of the part or all of amino acid sequence release of the AveC homologue gene outcome of purifying.
5.2. recombination system
5.2.1. cloning and expression carrier
The present invention further provides recombinant cloning vector and expression vector, it is of the present invention that it can be used for Cloning and Expression, includes the polynucleotide molecule of deinsectization streptomycete aveC ORF or arbitrary aveC homologue ORF. In a non-limiting embodiments, the invention provides plasmid pSE186 (ATCC 209604), it includes the ORF of deinsectization streptomycete aveC gene complete.
Unless stated otherwise, relevant deinsectization streptomycete aveC ORF or include the polynucleotide molecule of deinsectization streptomycete aveC ORF or its part below all, or the description of deinsectization streptomycete AveC gene product, also refer to AveC homologue and AveC homologue gene product simultaneously.
Develop the multiple different carrier that specifically is used for streptomycete, comprised phage, high copy number plasmid, low copy number plasmid, and intestinal bacteria-streptomycete shuttle plasmid etc., wherein any can be used for putting into practice the present invention.Also cloned the high amount of drug resistant gene from streptomycete, wherein some gene is impregnated in carrier as selected marker.At other document such as Hutchinson, 1980, applied biochemistry and biotechnology have been enumerated the example that uses these carriers in streptomycete among the 16:169-190.
Recombinant vectors of the present invention, the especially expression vector that preferably constructs make polynucleotide molecule of the present invention encoding sequence with transcribe or translate the necessary one or more controlling elements of encoding sequence and can operate and link to each other to produce polypeptide.Term used herein " controlling element " includes but not limited to such nucleotide sequence, derivable and the not derivable promotor of its coding, enhanser, operation, and other element that is used to drive and/or regulate and control the expression of polynucleotide encoding sequence known in the art.Simultaneously, as used herein, this encoding sequence " can be operated and link to each other " with one or more controlling elements, and wherein these controlling elements are regulated and allowed to transcribe encoding sequence effectively or translate its mRNA, has perhaps both transcribed also translation.
The typical plasmid vector that can be processed to include polynucleotide molecule of the present invention comprises pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), and shuttle vectors pWHM3 (Vara etc., 1989, the bacteriology magazine, 171:5872-5881) etc.
The method that structure includes the recombinant vectors of specific coding sequence (it can be operated with suitable controlling element and link to each other) is well-known in the art, can use these methods to put into practice the present invention.These methods comprise extracorporeal recombination, synthetic technology, vivo gene reorganization.Example is seen Maniatis etc., 1989, and document is the same; Ausubel etc., 1989, document is the same; Sambrook etc., 1989, document is the same; Innis etc., 1995, document is the same; And Erlich, 1992, the technology of describing during document is the same.
The intensity of the controlling element of these carriers can be different with specificity.According to used host/vector system, can use multiple suitable in the element any transcribed and translate.For bacterium, the non-limitative example of transcription regulatory region or promotor includes; β-gal promotor, the T7 promotor, the TAC promotor, a λ left side and right promotor, trp and lac promotor, the trp-lac promoter, fusion, then more special for streptomycete, promotor is ermE, melC, and tipA etc.In the described specific embodiment of the 11st part, an expression vector that includes aveC ORF is produced below, and described ORF is cloned in the strong composing type ermE promotor position adjacent with red saccharopolyspora.This carrier is transformed in the deinsectization streptomycete, shows that with HPLC analysis tunning the titre of the avermectin that is produced increases to some extent with respect to the output of only expressing the allelic identical bacterial strain of wild-type aveC subsequently.
Fusion protein expression vector can be used to express AveC gene product-fusion rotein.The fusion rotein of purifying can be used to improve the antiserum(antisera) of anti-AveC gene product, is used to study the biochemical characteristic of AveC gene product, with different biochemical activity processing AveC fusion rotein, or helps to identify and AveC gene product that purifying is expressed.Possible fusion protein expression vector is including, but not limited to such carrier, it is integrated with coding beta-galactosidase and trpE fusant, the maltose binding protein fusant, the sequence of glutathione-S-transferase fusant and polyhistidine fusant (delivery zone).In an alternative embodiment, AveC gene product or its part can with AveC homologue gene product or its meromixis, this AveC homologue gene product derives from other kind or the bacterial strain of streptomycete, for example streptomyces hygroscopicus or ash produce the look streptomycete.In the 12nd part and figure-7 described particular, made up chimeric plasmid below, it contains the zone of the 564bp of streptomyces hygroscopicus aveC homologue ORF, and it has substituted the zone of the homology 564bp of deinsectization streptomycete aveC ORF.This heterozygosis carrier is converted in the deinsectization streptomycete cell, and detects its avermectin 2 classes: the influence of the ratio of 1 class being produced.
The AveC fusion rotein can be designed to include the zone useful to purifying.For example, can use amylose resin purifying AveC-maltose binding protein fusant; Can use glutathione agarose pearl purifying AveC-glutathione-S-transferase fusion rotein; Can use nickelous resin purification AveC-polyhistidine fusant.Perhaps, the antibody of anti-carrier proteins or polypeptide can be used to the affinitive layer purification of fusion rotein.For example, can be with operating the appropriate position that the nucleotide sequence of the coding monoclonal antibody target epi-position that links to each other inserts expression vector, so that epi-position of expressing and the fusion of AveC polypeptide with controlling element.For example, can be by the standard technique FLAG that will encode TMThe nucleotide sequence of epi-position mark (InternationalBiotechnologies Inc.) (it is a wetting ability mark peptide) inserts in the expression vector site corresponding to AveC polypeptide C-terminal.Can utilize the anti-FLAG that is purchased TMThe AveC polypeptide FLAG that antibody test and affinity purification are expressed TMThe epi-position fusion product.
Coding AveC Expression of Fusion Protein carrier equally also can be designed to include the polylinker sequence of the special protease cracking site of coding, so that the AveC polypeptide of expressing discharges through can or merging the counterpart from the carrier zone with specific protease treatment.For example, this fusion rotein carrier can comprise the dna sequence dna of coding zymoplasm or Xa factor restriction enzyme site etc.
Utilize known method the signal sequence in aveC ORF upstream and the frame to be inserted the transportation and the secretion of the gene product of expressing with mediation in the expression vector.The non-limitative example of signal sequence comprises and derives from alpha factor, immunoglobulin (Ig), outer membrane protein, the signal sequence of penicillinase and TXi Baoshouti etc.
Transform or the host cell of transfection with clone of the present invention or expression vector in order to help to select, but this carrier also can be designed to further comprise the encoding sequence of reporter gene product or other selective marker.Preferred described encoding sequence can be operated with above-mentioned controlling element encoding sequence and link to each other.To the useful reporter gene of the present invention is well-known in the art, and it comprises those coding green fluorescent proteins, luciferase, the reporter gene of xylE and tyrosine oxidase etc.But the nucleotide sequence of coding selective marker is well-known in the art, and it comprises that those codings give antibiotics resistance, the metabolic antagonist resistance, or the sequence of the gene product of auxotroph demand is provided.These sequences comprise the erythromycin of for example encoding, the nucleotide sequence of resistances such as thiostrepton or kantlex.
5.2.2 the conversion of host cell
The present invention further provides transformed host cells, it contains polynucleotide molecule of the present invention or recombinant vectors, or by new bacterial strain of this host cell deutero-or clone.The host cell that can be used in the invention process is preferably streptomyces cell, but other prokaryotic cell prokaryocyte or eukaryotic cell equally also can be used.This transformed host cells generally comprises but is not limited to microorganism, for example uses recombinant phage dna, plasmid DNA, or the bacterium that transforms of cosmid DNA carrier, or the yeast that transforms with recombinant vectors etc.
Polynucleotide molecule of the present invention is desired to play a role in streptomyces cell, and it also can and be expressed purpose because of the clone and be converted in other bacterium or the eukaryotic cell.For example generally use coli strain,, can derive from American type culture collection (ATCC), Rockville, MD, USA (preserving number 31343), or commercially available (Stratagene) as DH5 α bacterial strain.Preferred eukaryotic host cell comprises yeast cell, but mammalian cell or insect cell also can effectively be used.
In one or more host cells that recombinant expression vector of the present invention is preferably transformed or transfection to homogeneous is basically cultivated.Generally, for example, pass through protoplast transformation according to known technology, calcium phosphate precipitation, calcium chloride is handled, micro-injection, electroporation, carry out transfection by contacting with recombinant virus, liposome-mediated transfection, the transfection of DEAE-dextran, transduction, engage, or microparticle bombardment imports expression vector in the host cell.Can select transformant by standard method, for example, select to express the selected marker relevant, as the cell of antibiotics resistance with above-mentioned recombinant vectors.
In case expression vector is imported host cell, can the standard technique by the detection of desired gene product confirm that the aveC encoding sequence has been integrated and maintained on the host chromosome or exist with the form of episome, described technology such as Southern hybridization analysis, restricted enzyme cutting analysis, pcr analysis comprises reverse transcriptase PCR (rt-PCR) or immunoassay.Contain and/or the host cell of express recombinant aveC encoding sequence can confirm that described method comprises by at least four kinds of known general methods any: (i) DNA-DNA, DNA-RNA, perhaps RNA-sense-rna hybridization; The (ii) existence of certification mark gene function; (iii) assess transcriptional level by the expression of measuring aveC specific mrna transcript in the host cell, and (iv) by immunoassay or AveC is bioactive exists (for example, the generation of the avermectin of specified proportion and amount is represented to have the AveC activity in the deinsectization streptomycete host cell) to detect the existence of ripe polypeptide product.
5.2.3. The expression and the evaluation of heavy thin AveC gene product
In case the aveC encoding sequence is stabilized the importing proper host cell, this transformed host cells can be cultivated the gained cell by asexual reproduction under the condition that helps AveC gene product maximum to produce.Described condition generally comprises cell cultures to high-density.When expression vector contains inducible promoter, when needing abduction delivering, for example can use suitable inductive condition, temperature variation, nutrition exhausts, (for example add gratuitous inducer, the analogue of carbohydrate, for example isopropyl-(IPTG)), the accumulation of excessive metabolic by-prods etc.
When the AveC gene product of expressing is retained in host cell inside, collecting cell and cracking, under extraction conditions known in the art, for example, at 4 ℃ or the existence of proteinase inhibitor is arranged, or under the condition of the two coexistence, separation and purified product from lysate are so that proteinic minimum degradation.When the AveC gene product of expressing is secreted, only need to collect the nutritional medium that exhausts and separated product therefrom from host cell.
Utilize standard method, can from cell lysate or substratum (suitably time), separate or the AveC gene product of purifying expression basically, described method is including, but not limited to any combination of following method: ammonium sulfate precipitation, size fractionation separates, ion exchange chromatography, HPLC, density centrifugation and affinity chromatography.When the AveC gene product of expressing shows biological activity, can use suitable detection method to monitor the raising of goods purity in each step of purification process.No matter whether the AveC gene product of expressing shows biological activity, can be according to its size, with antibody or be specific to the reactivity of the antibody of AveC, perhaps merge mark in the presence of detect this gene product.
Therefore, the invention provides recombinant expressed deinsectization streptomycete AveC gene product and homologue thereof, described gene product contains can be by the coded aminoacid sequence of AveC gene product encoding sequence of plasmid pSE186 (ATCC 209604), or aminoacid sequence or its substantive part shown in Fig. 1 (SEQ ID NO:2).
The present invention further provides recombinant expressed streptomyces hygroscopicus AveC homologue gene product and homologue thereof, described gene product contains just like the aminoacid sequence shown in the SEQ ID NO:4 or its substantive part.
The present invention further provides the method for producing the AveC gene product, described method is included in and helps to produce under the condition of reorganization AveC gene product, cultivate and use the recombinant expression vector transformed host cells, and from cell culture, reclaim the AveC gene product, wherein said carrier contains the polynucleotide molecule of the nucleotide sequence with coding AveC gene product, and described polynucleotide molecule can be operated with the controlling element of the expression of one or more control polynucleotide molecules in host cell and link to each other.
Recombinant expressed deinsectization streptomycete AveC gene product can be used for different purposes, can change the compound of AveC gene product function comprising screening, and therefore modulates the biosynthesizing of avermectin and produce antibody at the AveC gene product.
In case obtain the sufficiently high AveC gene product of purity, just can the identified gene product by following standard method, described method such as SDS-PAGE, size exclusion chromatography, amino acid sequence analysis produces biological activity of suitable product or the like in the avermectin biosynthetic pathway.For example, can use the aminoacid sequence of the peptide sequencing technical measurement AveC gene product of standard.Can be with hydrophilicity analysis (example be seen Hopp and Woods, 1981, Proc.Natl.Acad.Sci.USA 78:3824), perhaps similarly software algorithm is identified the hydrophilic area and the hydrophobic region of AveC gene product, takes this further to identify the AveC gene product.Can carry out structural analysis to identify the zone that has special secondary structure in the AveC gene product, for example can use biophysics method (Engstrom as the X-radiocrystallgraphy, 1974, Biochem.Exp.Biol.11:7-13), computer model manufacture method (Fletterick and Zoller (volume), 1986, the up-to-date communication of molecular biology, cold spring harbor laboratory, cold spring port, New York) and nucleus magnetic resonance (NMR) mapping and research AveC gene product and substrate thereof between interactional site.The information that obtains by institute can be used to select the new mutant site of aveC ORF, has the more suitably new bacterial strain of deinsectization streptomycete of avermectin production characteristic to help exploitation.
5.3. The structure of AveC mutant and application
The present invention further provides polynucleotide molecule, itself or have the identical nucleotide sequence of deinsectization streptomycete AveC gene product encoding sequence with plasmid pSE186 (ATCC209604), or has the nucleotide sequence of the deinsectization streptomycete aveC ORF shown in Fig. 1 (SEQ ID NO:1), but it further includes one or more sudden changes so that deinsectization streptomycete bacterial strain ATCC 53692 (wherein wild-type aveC allelotrope inactivation and express the polynucleotide molecule of the nucleotide sequence that contains sudden change) cell of more only expressing the allelic deinsectization streptomycete bacterial strain ATCC 53692 of wild-type aveC produced the avermectin of different ratios and amount.
According to the present invention, described polynucleotide molecule can be used to produce the new bacterial strain of deinsectization streptomycete, described new bacterial strain with only express the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC relatively, the change that can survey takes place in the production of avermectin.In preferred embodiments, described polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (it is compared with only expressing the allelic identical bacterial strain of wild-type aveC, and with 2 classes that reduce: 1 class ratio is produced avermectin).In another preferred embodiment, described polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (its with only express the allelic identical bacterial strain of wild-type aveC compare the avermectin that can produce the increase level).In a further preferred embodiment, described polynucleotide molecule can be used for producing the new bacterial strain of deinsectization streptomycete (wherein aveC gene inactivation).
The sudden change of AveC encoding sequence comprises aminoacid deletion, adds or replaces to import the AveC gene product, or any sudden change that causes the brachymemma of AveC gene product or its any combination and produce required result.For example, the invention provides polynucleotide molecule, it comprises AveC gene product encoding sequence or the deinsectization streptomycete aveCORF nucleotide sequence shown in Fig. 1 (SEQ ID NO:1) of plasmid pSE186 (ATCC 209604), but it further includes one or more sudden changes, the replacement that its coding carries out amino-acid residue in the selected site of AveC gene product with different amino-acid residues.In the embodiment of the several non-limiting that exemplifies hereinafter, this replacement occurs in amino-acid residue the 55th, 138,139, or 230, or wherein several site.
Can carry out the sudden change of aveC encoding sequence by any of multiple currently known methods, comprise the use error-prone PCR, or pass through cassette mutagenesis.For example, can use oligonucleotide mediated mutagenesis for example to import one or more restriction sites or terminator codon changes aveC ORF sequence in the specific region of aveC ORF sequence in a kind of definite mode.As United States Patent (USP) 5,605,793 is described, also can use to comprise segmentization at random, and the method for repetition mutagenesis circulation and Nucleotide reorganization produces the polynucleotide library of the bigger nucleotide sequence with coding aveC sudden change.
The target sudden change is useful, is particularly useful for the one or more conservative amino-acid residue that changes the AveC gene product.For example, as shown in Figure 6, compare by the amino acid residue sequence that AveC gene product and the AveC homologue gene product of producing look streptomycete (SEQ ID NO:5) and streptomyces hygroscopicus (SEQ ID NO:4) from deinsectization streptomycete (SEQ ID NO:2), ash are inferred, shown amino-acid residue site significantly conservative between these kinds.The targeted mutagenesis that causes one or more described conservative amino-acid residues to change can show that for producing the new mutant strain that required disinsection mychel yield changes is very effective.
Random mutagenesis also is useful, can by with the deinsectization streptomycete cellular exposure in ultraviolet ray or x-ray irradiation, perhaps be exposed to as N-methyl-N '-nitrosoguanidine, the methanesulfonic ethyl ester, the chemical mutagen of nitrous acid or mustargen subclass carries out mutagenesis.The summary of relevant induced-mutation technique can be referring to Ausubel, and 1989, document is the same.
In case produce the polynucleotide molecule of sudden change, can screen to measure it whether to modulate avermectin biosynthesizing in the deinsectization streptomycete.In preferred embodiments, by complementary deinsectization streptomycete bacterial strain (thereby wherein the aveC gene inactivation provide the aveC-background) detect the polynucleotide molecule of nucleotide sequence with sudden change.In non-limiting method, the polynucleotide molecule of sudden change is become can operate the expression plasmid that links to each other with one or more controlling elements by montage, and wherein plasmid preferably includes one or more drug resistance genes to select transformant.Use known technology that this carrier is converted into the aveC-host cell, the selection transformant is also cultivated in suitable fermention medium under the condition that allows or induce avermectin to produce.Analyze tunning to measure the ability of the complementary host cell of polynucleotide molecule that suddenlys change by HPLC then.8.3 parts will illustrate to carry and can reduce avermectin B2 below: several carriers of the polynucleotide molecule of the sudden change of B1 ratio, it comprises pSE188, pSE199 and pSE231.
The invention provides the method for identifying deinsectization streptomycete aveC ORF sudden change (it can change the ratio and/or the output of the avermectin that is produced).In preferred embodiments, the invention provides and identify that (it can change the B2 of the avermectin that is produced: the method B1 ratio) in deinsectization streptomycete aveC ORF sudden change, described method comprises: (a) measure avermectin 2 classes that the deinsectization streptomycete bacterial strain cell produces: the ratio of 1 class, wild-type aveC allelotrope in the described cell is inactivation, and the polynucleotide molecule of nucleotide sequence that includes the AveC gene product of encoding mutant is imported into this cell and is expressed therein; (b) measure avermectin 2 classes of the strain cell generation identical with the deinsectization streptomycete of step (a): the ratio of 1 class, but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ ID NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; Reach (c) avermectin 2 classes of the deinsectization streptomycete bacterial strain cell generation of comparison step (a): avermectin 2 classes that the deinsectization streptomycete bacterial strain cell of the ratio of 1 class and step (b) produces: the ratio of 1 class, if the two difference then identifies and can change avermectin 2 classes: the aveC ORF sudden change of 1 class ratio exists.In preferred embodiments, avermectin 2 classes: the ratio of 1 class reduces because of sudden change.
In another preferred embodiment, the invention provides a kind of method, the gene construct that this method is used for identifying aveC ORF or include aveC 0RF can change the sudden change of the avermectin amount that is produced, described method comprises: the amount of (a) measuring the avermectin of deinsectization streptomycete bacterial strain cell generation, wild-type aveC allelotrope in the described cell is inactivation, includes the nucleotide sequence of AveC gene product of encoding mutant or the polynucleotide molecule of gene construct that contains the nucleotide sequence of coding AveC gene product and is imported into this cell and expresses therein; (b) measure the amount of the avermectin of the strain cell generation identical with the deinsectization streptomycete of step (a), but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ ID NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; And (c) amount of amount and the avermectin of the deinsectization streptomycete bacterial strain cell generation of step (b) of the avermectin that produces of the deinsectization streptomycete bacterial strain cell of comparison step (a), if the two difference then identifies the aveC ORF sudden change or the gene construct sudden change that can change the avermectin amount and exists.In preferred embodiments, the amount of avermectin increases because of sudden change.
Above-mentioned arbitraryly be used to identify that the method for sudden change can be undertaken by using fermention medium, preferably in this substratum, add hexahydrobenzoic acid, but other suitable fatty acids precursor, for example, also can be used as in the listed lipid acid precursor of table 1 any.
In case identify the polynucleotide molecule of modulating the sudden change that avermectin produces in the desired direction, the position of undergoing mutation on also can the definite kernel nucleotide sequence.For example, can by PCR separate the AveC gene product contain encoding mutant nucleotide sequence polynucleotide molecule and use currently known methods that it is carried out dna sequence analysis.By the allelic dna sequence dna of aveC relatively sudden change and wild-type, can determine to be responsible for changing the sudden change that avermectin is produced.The present invention concrete but in the nonrestrictive embodiment, contain the 55th (S55F), 138 (S138T), 139 (A139T) or 230 (G230D) position residue single amino acids replaces or the AveC gene product function of the deinsectization streptomycete AveC gene product of the 138th (S138T) and the dual replacements of 139 (A139T) position residue changes, and makes avermectin 2 classes that produced: the ratio of 1 class change (the 8th part vide infra).Therefore, the present invention includes polynucleotide molecule, the deinsectization streptomycete AveC gene product of the sudden change that its nucleotide sequence that contains is coded comprises the 55th, 138, one or more aminoacid replacement that 139 or 230 amino acids residues or its arbitrary combination place take place.
The present invention further provides the composition for preparing new deinsectization streptomycete bacterial strain, the cell of this bacterial strain contains the aveC allelotrope of sudden change and the variation that causes avermectin to be produced.For example, the invention provides recombinant vectors, can use this carrier with the aveC gene locus on any polynucleotide molecule target deinsectization streptomycete karyomit(e) that contains sudden change nucleotide sequence of the present invention, to insert by homologous recombination or to replace aveC ORF or its part.Yet, according to the present invention, the polynucleotide molecule of the nucleotide sequence that contains the present invention's sudden change provided herein also can be inserted on the deinsectization streptomycete karyomit(e) except that the extragenic site of aveC, or when in the deinsectization streptomycete cell, keeping unbound state, play the biosynthetic effect of modulation avermectin equally.Therefore, the present invention also provides the carrier of the polynucleotide molecule with the nucleotide sequence that contains the present invention's sudden change, can use these carriers that polynucleotide molecule is inserted into the position except that the aveC gene locus on the deinsectization streptomycete karyomit(e), or keep unbound state.
In preferred embodiments, the invention provides the gene replacement vector, it can be used for the aveC allelotrope of sudden change is inserted deinsectization streptomycete bacterial strain, take this to produce new deinsectization streptomycete bacterial strain, the cell of described bacterial strain is compared with the allelic identical strain cell of the aveC that only expresses wild-type, and avermectin 2 classes of generation: 1 class ratio changes to some extent.In preferred embodiments, avermectin 2 classes of this cell generation: 1 class ratio reduces.Can use expression vector provided herein such as pSE188, pSE199, and the polynucleotide molecule of the sudden change that exists among the pSE231 (these expression vectors the 8.3rd part below will illustrate) makes up described gene replacement vector.
In a further preferred embodiment, the invention provides carrier, can use this carrier that the aveC allelotrope of sudden change is inserted in the deinsectization streptomycete bacterial strain cell to produce new cell strain, described new cell is compared with the allelic identical strain cell of the AveC that only expresses wild-type, and the amount of the avermectin of generation changes to some extent.In preferred embodiments, the amount of the avermectin of described new cell generation increases.In concrete, non-limiting embodiments, this carrier further comprises strong promoter known in the art, for example, from the strong composing type ermE promotor of Saccharopolyspora erythraea, it is positioned at aveC ORF upstream and can operates with aveC ORF and links to each other.Described carrier can be as following embodiment 11 described plasmid pSE189, and the aveC allelotrope of sudden change that maybe can be by using plasmid pSE189 makes up.
In a further preferred embodiment, the invention provides the gene replacement vector, this carrier can be used for the aveC gene in the deactivation wild-type deinsectization streptomycete bacterial strain.In nonrestrictive embodiment, can make up described gene replacement vector by the polynucleotide molecule that uses the sudden change that exists among the plasmid pSE180 (ATCC 209605), its below the 8.1st part illustrate (Fig. 3).The present invention further provides the gene replacement vector that includes polynucleotide molecule, described polynucleotide molecule comprises the nucleotide sequence of natural flank original position aveC gene on deinsectization streptomycete karyomit(e) or is made up of this sequence, for example, comprise those flanking nucleotide sequences shown in Fig. 1 (SEQ ID NO:1), can use described carrier disappearance deinsectization streptomycete aveC ORF.
The present invention further provides the method for preparing the new bacterial strain of deinsectization streptomycete, described new bacterial strain contains the allelic cell of aveC of expressing sudden change, it compares with the cell of only expressing the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC, and the ratio and/or the amount of the avermectin that is produced change.In preferred embodiments, the present invention further provides the method for preparing the new bacterial strain of deinsectization streptomycete, described new bacterial strain contains the allelic cell of aveC of expressing sudden change, it compares with the cell of only expressing the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC, avermectin 2 classes that produced: 1 class ratio changes, described method comprises with the allelic carrier conversion of the aveC that carries sudden change deinsectization streptomycete bacterial strain cell, the gene product of the aveC allelotrope coding of this sudden change makes: compare with the cell of only expressing the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC, 2 classes of the avermectin that the allelic deinsectization streptomycete bacterial strain cell of aveC of expression sudden change produces: 1 class ratio changes to some extent; Avermectin 2 classes that selection is produced: the ratio of 1 class is more only expressed the transformant that the allelic strain cell of wild-type aveC changes to some extent.In preferred embodiments, avermectin 2 classes: the ratio of 1 class reduces to some extent.
In a further preferred embodiment, the invention provides the method for preparing the new bacterial strain of deinsectization streptomycete, described bacterial strain contains the cell of the avermectin of production change amount, described method comprises with carrying suddenlys change the allelic carrier of aveC or contains the allelic gene construct conversion of this aveC deinsectization streptomycete bacterial strain cell, and the amount that the allelic expression of the aveC of described sudden change makes the amount of the avermectin that the deinsectization streptomycete bacterial strain cell of expression sudden change aveC allelotrope or gene construct is produced more only express the avermectin of the identical strain cell generation of the allelic deinsectization streptomycete of wild-type aveC changes to some extent; The amount of the avermectin that selection is produced is more only expressed the transformant that the allelic strain cell of wild-type aveC changes to some extent.In preferred embodiments, the amount of the avermectin that is produced in the transformant increases to some extent.
In a further preferred embodiment, the invention provides the method for preparing the new bacterial strain of deinsectization streptomycete, the cell of described bacterial strain contains the aveC allelotrope of inactivation, and described method comprises with the allelic carrier conversion of the aveC of inactivation expresses the allelic deinsectization streptomycete bacterial strain cell of wild-type aveC; Select wherein the aveC allelotrope transformant of inactivation.In preferred, nonrestrictive embodiment, the deinsectization streptomycete bacterial strain cell is transformed by the gene replacement vector, the entrained aveC allelotrope of this carrier replaces the allelic part inactivation of AveC, the transformant of wherein selecting natural aveC allelotrope to be replaced by the aveC allelotrope of inactivation because of sudden change or with the heterologous gene sequence.As described below, the allelic inactivation of AveC can be measured by analyzing tunning with HPLC.As below in described concrete, the nonrestrictive embodiment of the 8.1st part, make the aveC allelic inactivation by the ermE gene that in aveC ORF, inserts from red saccharopolyspora.
The present invention further provides the new bacterial strain of deinsectization streptomycete, it contains useful arbitrary polynucleotide molecule of the present invention or carrier cell transformed.In preferred embodiments, the invention provides the new bacterial strain of deinsectization streptomycete, it contains the aveC allelotrope of expressing sudden change with the aveC allelotrope that replaces wild-type or contain and express this two kinds of allelic cells simultaneously, and 2 classes of wherein new strain cell to change to some extent for the allelic identical strain cell of the aveC that only expresses wild-type: the ratio of 1 class is produced avermectin.In preferred embodiments, 2 classes of the avermectin of new cell generation: 1 class ratio reduces.This new bacterial strain is very useful for the scale operation of commercial avermectin such as doramectin.
The basic goal of shaker test described herein is to identify the aveC allelotrope of sudden change, and its expression in the deinsectization streptomycete cell has changed, and has particularly reduced by 2 classes of the avermectin that produces: the ratio of 1 class.In preferred embodiments, expression of the present invention can reduce by 2 classes of the avermectin that is produced: the B2 of the avermectin that the new strain cell of the allelic deinsectization streptomycete of the aveC of the sudden change of 1 class ratio is produced: the scope that the ratio of B1 reduces be 1.6: 1～below 0: 1; In a more preferred embodiment, ratio be about 1: 1～0: 1; In the most preferred embodiment, ratio is about 0.84: 1～and 0: 1.In the specific embodiments that is described below, the cyclohexyl B2 that new cell of the present invention produced: the ratio of cyclohexyl B1 avermectin was less than 1.6: 1.In the different specific embodiments of another that is described below, the cyclohexyl B2 that new cell of the present invention produced: the ratio of cyclohexyl B1 avermectin approximately is 0.94: 1.In the different specific embodiments of another that is described below, the cyclohexyl B2 that new cell of the present invention produced: the ratio of cyclohexyl B1 avermectin approximately is 0.88: 1.In the different specific embodiments of another that is described below, the cyclohexyl B2 that new cell of the present invention produced: the ratio of cyclohexyl B1 avermectin approximately is 0.84: 1.
In a more preferred embodiment, the invention provides the new bacterial strain of deinsectization streptomycete, it contains the aveC allelotrope of expressing sudden change, contain the allelic gene construct of aveC with replace wild-type aveC allelotrope or contain express sudden change simultaneously with the allelic cell of wild-type aveC, wherein new strain cell is produced avermectin with the amount that changes to some extent for only expressing the allelic identical strain cell of wild-type aveC.In preferred embodiments, the amount of the avermectin of new bacterial strain generation increases.In nonrestrictive embodiment, gene construct further comprises strong promoter, and for example, from the strong composing type ermE promotor of red saccharopolyspora, it is positioned at aveC ORF upstream and can operates with aveC ORF and links to each other.
In a further preferred embodiment, the invention provides the new bacterial strain of deinsectization streptomycete, it contains wherein the aveC gene cell of inactivation.Described bacterial strain can be used for producing with wild type strain compares the avermectin that different spectrums are arranged, and also can measure the production whether aveC gene target or random mutagenesis influence avermectin in complementary shaker test as herein described.In the described hereinafter specific embodiments, the deinsectization streptomycete host cell contains the aveC gene of inactivation after genetic modification.For example, below the bacterial strain SE180-11 described in the embodiment just be to use gene to replace that plasmid pSE180 (ATCC209605) (Fig. 3) produces, by making deinsectization streptomycete aveC gene inactivation at aveC gene coding region insertion ermE resistant gene.
The present invention further provides recombinant expressed, deinsectization streptomycete AveC gene product of sudden change and preparation method thereof, described gene product is by any coding of the above-mentioned polynucleotide molecule of the present invention.
The present invention further provides the method for producing avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim described avermectin, the aveC allelotrope of the sudden change of described cell expressing encoding gene product, compare with the allelic identical strain cell of the aveC that only expresses wild-type, the allelotrope of described sudden change has changed 2 classes of the avermectin of the allelic deinsectization streptomycete bacterial strain cell generation of the aveC that expresses sudden change: the ratio of 1 class.In preferred embodiments, express 2 classes of the avermectin of the allelic cell generation of the aveC that suddenlys change: the ratio of 1 class reduces to some extent.The avermectin that this method provides commercially valuable is the high efficiency production method of doramectin for example.
The present invention further provides the method for producing avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim described avermectin, the aveC allelotrope of described cell expressing sudden change or contain the allelic gene construct of aveC, only express the allelic identical strain cell of aveC of wild-type with not expressing sudden change aveC allelotrope or gene construct and compare, the amount of the avermectin that the aveC allelotrope of expression sudden change or the deinsectization streptomycete bacterial strain cell of gene construct produce changes to some extent.In preferred embodiments, the amount of the avermectin that the cell of expression sudden change aveC allelotrope or gene construct produces in culture increases to some extent.
The present invention further provides the new composition of the avermectin that produces by the allelic deinsectization streptomycete bacterial strain of the aveC that expresses sudden change, wherein, avermectin 2 classes that the coded gene product of aveC allelotrope of sudden change is produced the allelic deinsectization streptomycete bacterial strain cell of aveC of expression sudden change: the ratio of 1 class is compared with the cell of only expressing the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC and is decreased, wherein, avermectin 2 classes with the cell generation of only expressing the identical bacterial strain of the allelic deinsectization streptomycete of wild-type aveC: 1 class ratio is compared, and 2 classes of the avermectin in the novel composition to reduce: 1 class ratio produces.This novel avermectin composition may reside in the fermentation culture that exhausts or therefrom reclaims, and can be by known biochemical purification technology such as ammonium sulfate precipitation, dialysis, size fractionation separates, ion exchange chromatography, HPLC etc. be partial purification or basic purifying from nutrient solution.
5.4. the purposes of avermectin
Avermectin is a kind of very effective antiparasitic agents, particularly kills medicine, sterilant and miticide as wormer, epizoon.All can be used for these purposes according to the avermectin compound that method of the present invention produced.For example, can be used for treating human various diseases or symptom, those diseases that cause by parasitic infection especially known in the art according to the avermectin compound that method of the present invention produced.Referring to Ikeda and Omura, 1997, Chem.Rev.97 (7): 2591-2609.More specifically be that for producing effective therapeutic action by various diseases or symptom that entozoa caused, described entozoa for example parasitic nematode class can infect human body according to avermectin compound that method of the present invention produced, domestic animal, pig, sheep, poultry, horse or ox.
More particularly, infect the nematode of human body according to avermectin compound that method of the present invention produced for opposing and infect the nematodes of various animals all effective.Such nematode comprises stomach parasite such as hookworm, nematode, roundworm, quasi-colubriformis, Trichinella spiralis, capillaria, whipworm, pinworm, the enteron aisle extract of dirofilariasis and the parasite of in blood vessel, other tissue or organ, finding such as filaria property worm and class garden genus and Trichinella spiralis.
Also can be used for treating epizoon according to the avermectin compound that method of the present invention produced and infect, for example comprise that the segmental appendage that moves mammals that the Diptera larvae of dwelling etc. causes or birds etc. by spiral shell, mite, louse, flea, blow fly, gallinipper or harassing and wrecking horse, ox infects.
Also can be used as domestic pesticide according to the avermectin compound that method of the present invention produced, as kill cockroach, casemaking clothes moth, carpet beetle, and housefly etc., and, comprise gamasid, aphid, caterpillar and lepidopterans larva such as locust etc. to storage of granular materials and the deleterious insect of farm crop.
The animal of using the avermectin compound that produces according to method of the present invention to treat comprises that sheep, ox, horse, deer, goat, pig, birds comprise poultry, dog and cat.
Can be mixed with the formulation that is suitable for specific end use according to the avermectin compound that method of the present invention produced, this is with relevant by the particular types of the host animal of being treated, related parasite and insect.For using as a kind of wormer, can be oral according to the avermectin compound that method of the present invention produced with the form of capsule, pill, tablet, liquid Haust for animals, perhaps to pour into, injection or the form of implanting are carried out administration.This preparation can be put into practice by the veterinary science of standard in a conventional manner and be prepared.Like this, capsule, pill or tablet can contain disintegrating agent and/or tackiness agent such as starch, lactose, talcum, Magnesium Stearate etc. in addition by activeconstituents is mixed with suitable purified diluent or carrier.The preparation of Haust for animals is that activeconstituents is dispersed in the aqueous solution of dispersion agent or wetting agent etc.Injection can be mixed with thimerosal, wherein can contain other material such as enough salt and/or glucose oozes this solution and blood etc.
Relevant absorption of active ingredient changes according to the difference of the seriousness of the kind of patient, the host animal of being treated, infection and type, host's body weight etc. in these preparations.In general, for the preparation of oral administration, the dosage of active ingredient can all can with single dose or administered in divided doses in 1 day to 5 days from 0.001 body weight to 10mg/kg patient or animal.Yet in some cases, physician or animal doctor can give higher or lower dosage according to clinical symptom.
Perhaps, also can for this purpose, also spissated food additive or pre-mixture can have been mixed in the animal-feed according to the avermectin compound that method of the present invention produced with the animal-feed administration.For as a kind of sterilant and handle Agricultural pests, also can be according to the avermectin compound that method of the present invention produced with spraying, dusting, emulsion and similar fashion are used by the method for standard on the agricultural.
6. Embodiment: the analysis of the fermentation of deinsectization streptomycete and avermectin B2: B1
Do not replenish lipid acid if lack the bacterial strain of side chain 2-oxygen acid-desaturase and 5-0-methylferase active substance simultaneously in fermentation media, it will not produce avermectin.This embodiment has proved in such mutant strain, when causing biosynthesizing in the presence of different lipid acid are arranged, can obtain the very wide avermectin of proportional range of B2: B1.
6.1. Material and method
Deinsectization streptomycete ATCC 53692 is stored in-70 ℃ the full meat soup seed culture medium, the consisting of of substratum: starch (Nadex, Laing National)-20g; The medicine medium (Trader ' s Protein, Memphis, TN)-15g; Ardamine pH (YeastProducts Inc.)-5g; Lime carbonate-1g.The volume final with the tap water adjustment was 1 liter, and pH is adjusted to 7.2,121 ℃ of following autoclavings 25 minutes.
The suspension that thaws of getting the above-mentioned preparation of 2ml is inoculated in the flask that contains the same substratum of 50ml.After on 28 ℃ of gyrate shakers it being cultivated 48 hours at 180rpm, be inoculated in the flask that contains 50ml production substratum with 2ml meat soup, this substratum contains: starch-80g: lime carbonate-7g; Pharmamedia-5g; Dipotassium hydrogen phosphate-1g; Sal epsom-1g; L-glutamic acid-0.6g; Ferrous sulfate-0.01g; Zinc sulfate-0.001g; Inferior magnesium-the 0.001g of sulfuric acid.The volume final with the tap water adjustment was 1 liter, and pH is adjusted to 7.2,121 ℃ of following autoclavings 25 minutes.
Different carboxylic acid enzyme substratess (referring to table 1) in methyl alcohol, dissolve and add in the meat soup base of fermentation inoculation after 24 hours ultimate density be 0.2g/1.The broth culture of fermentation was cultivated 14 days at 28 ℃, then with its centrifugal (2,500rpm, 2 minutes), removed supernatant liquor.With acetone (15ml), use methylene dichloride (30ml) extraction mycelia precipitation then, separate organic phase, filter, then evaporation drying.Resistates absorbs with 1ml methyl alcohol, and carry out HPLC with the Hewlett-Packard 1090A liquid chromatography of the scanning diode group detector of being furnished with 240nm and analyze, used pillar is BeckmanUltrasphere C-18,5 μ m, 4.6mm the pillar of * 25cm remains on 40 ℃.Above-mentioned methanol solution 25 μ m are injected pillar.Under 0.85/ milliliter minute, carried out wash-out from 80: 20 to 95: 5 above 40 minutes with the methanol-water linear gradient.Two kinds of normal concentrations of cyclohexyl B1 are used for the reaction of calibrated detector, measure the area corresponding to the curve of avermectin B2 and B1.
6.2. The result
The HPLC retention time of viewed avermectin B2 and B1, and 2: 1 ratios are as shown in table 1.
Table 1
The HPLC retention time (minute) Ratio
Substrate B2 ?B1 ?B2∶B1
The 4-tetrahydropyran carboxylic acid 8.1 ?14.5 ?0.25
Isopropylformic acid 10.8 ?18.9 ?0.5
The 3-furancarboxylic acid 7.6 ?14.6 ?0.62
S-(+)-2-Methyl Butyric Acid 12.8 ?21.6 ?1.0
Hexahydrobenzoic acid 16.9 ?26.0 ?1.6
The 3-thiophene carboxylic acid 8.8 ?16.0 ?1.8
Cyclopentane carboxylic acid 14.2 ?23.0 ?2.0
3-trifluoromethyl butyric acid 10.9 ?18.8 ?3.9
2 methyl valeric acid 14.5 ?24.9 ?4.2
The Cycloheptanoic acid 18.6 ?29.0 ?15.0
The listed digital proof of table 1 B2 of the avermectin that produces: the ratio of B1 has a very wide scope, has shown that the result of 2 compounds and 1 compounds dehydration conversion has very big difference, and character of the fatty acid side chain start unit that is provided is provided for this.This shows B2: the change of B1 ratio (being produced by the proteic change of aveC) has specificity for specific substrate.Therefore, the displaying B2 that obtains with specific substrate: the screening of the muton that the B1 ratio changes also need be carried out in the presence of this substrate.Below these examples described the use hexahydrobenzoic acid as the screening substrate.Yet this substrate only is used to illustrate potential practicality of the present invention, rather than is meant to limit the present invention.
7. Embodiment: the separation of aveC gene
Present embodiment has been described the separation and the evaluation in the deinsectization streptomycete karyomit(e) zone of coding AveC gene prod, and as described down, the aveC gene is accredited as and can changes the avermectin cyclohexyl B2 that produced and the ratio (B2: B1) of B1.
7.1. Materials and methods
7.1.1. Be used for the growth of the isolating streptomycete of DNA
Following method is used to cultivate streptomycete.The single bacterium colonies of deinsectization streptomycete ATCC 31272 strains (single bacterium colony isolate #2) separate in the YPD-6 of 1/2 concentration, and YPD-6 includes: Difco YeastExtract-5g; Difco Bacto-peptone-5g; Glucose-2.5g; MOPS-5g; Difco Bacto agar-15g., final volume dH 2O is adjusted to 1 liter, and pH is adjusted to 7.0, and substratum was 121 ℃ of following autoclavings 25 minutes.
The mycelium of above-mentioned culture medium culturing is used to be inoculated into 10ml TSB substratum, and (DifcoTryptic Soy Broth-30g is at 1 liter of dH 2Among the O, 121 ℃ of following autoclavings 25 minutes), in a 25mm * 150mm test tube,, cultivated 48-72 hour down at 28 ℃ with the 300rpm vibration.
7.1.2. From streptomycete, separate chromosomal DNA
The five equilibrium mycelium (0.25ml or 0.5ml) of above-mentioned growth is positioned over the microcentrifugation test tube of a 1.5ml and this cell is concentrated centrifugal 60 seconds of 12,000 * g.Remove supernatant liquor, make cell suspension (20ml 1.5M sucrose, 2.5ml 1MTris-HCl, pH8.0,2.5ml 1M EDTA, pH8.0 and 75ml dH in 0.25ml TSE damping fluid 2O), it contains the 2mg/ml N,O-Diacetylmuramidase.Sample was 37 ℃ of following shaking culture 20 minutes, and delivery is to an AutoGen540 TM(MA), genomic dna separates according to operational manual with Cycle 159 (instrument software) the separate nucleic acid device for Integrated Separation Systems, Natick automatically.
Perhaps, the mycelium of 5ml is placed the test tube of a 17mm * 100mm,, carried out cell concentration in centrifugal 5 minutes under the 000rpm, remove supernatant liquor by 3.Cell is suspended in the 1ml TSE damping fluid again, by 3, carries out cell concentration in centrifugal 5 minutes under the 000rpm, removes supernatant liquor.Cell is suspended in the TSE damping fluid that 1ml contains the 2mg/ml N,O-Diacetylmuramidase again, and hatches 30-60 minute 37 ℃ of vibrations.After cultivating, add 0.5ml 10% sodium lauryl sulphate (SDS), cell is hatched under 37 ℃, finish up to cracking.Split product was hatched under 60 ℃ 10 minutes, was cooled to room temperature, assign in two 1.5ml Eppendorf test tubes, and (50% phenol was used 0.5M Tris balance, pH8.0 in advance with the 0.5ml phenol/chloroform; 50% chloroform) extraction is 1 time.Remove water, use chloroform: primary isoamyl alcohol (24: 1) extraction 2 to 5 times.By adding 1/10 volume 3M sodium-acetate, the pH4.8 prep dna, mixtures incubated is 10 minutes in ice, 15,000rpm, 5 ℃ with centrifugal 10 minutes of mixture, supernatant liquor is moved to a clean test tube, and add the Virahol of 1 volume therein.Then supernatant liquor was hatched in ice 20 minutes with Virahol, 15,000rpm, 5 ℃ with centrifugal 20 minutes of mixture, supernatant liquor is removed, with 70% washing with alcohol DNA granular sludge 1 time.After the granular sludge drying, with the DNA resuspending in the TE damping fluid (10mM Tris, 1mM EDTA, pH8.0).
7.1.3. Isolated plasmid dna from streptomycete
A part of mycelium (1.0ml) is positioned over the microcentrifugation test tube of a 1.5ml, and it is 12, centrifugal 60 seconds concentrating cells under the 000xg.Remove supernatant liquor, the cell resuspending centrifugally under 12,000 * g concentrates 60 seconds in 1.0ml10.3% sucrose, remove supernatant liquor.Cell is resuspended in the 0.25ml TSE damping fluid then, it contains the 2mg/ml N,O-Diacetylmuramidase, vibrates down at 37 ℃ and hatches 20 minutes, is loaded onto AutoGen 540 then TMAutomatically in the separate nucleic acid device.According to operational manual, use Cycle 106 (instrument software) isolated plasmid dna.
Perhaps, the mycelium of 1.5ml is positioned in the miniature centrifuge tube of 1.5ml, 12, centrifugal 60 seconds concentrating cells under the 000xg.Remove supernatant liquor, the cell resuspending is in 1.0ml 10.3% sucrose, centrifugally under 12,000 * g concentrates 60 seconds, removes supernatant liquor.Cell is resuspended in the 0.5ml TSE damping fluid, and it contains the 2mg/ml N,O-Diacetylmuramidase, hatches 15-30 minute at 37 ℃.After hatching, (0.3N NaOH 2%SDS), was hatched under 55 ℃ 15-30 minute or clear up to solution becomes to add 0.25ml alkalescence SDS.(0.1ml, 3M pH4.8) join in the dna solution, and it was hatched in ice 10 minutes with sodium-acetate.The DNA sample under 5 ℃ 14, centrifugal 10 minutes of 000rpm.Supernatant liquor is moved to a clean test tube, and add phenol/chloroform (50% phenol: 50% chloroform) and gently mix of 0.2ml therein.With 14, centrifugal 10 minutes of 000rpm, supernatant liquor move to a clean Eppendorf test tube to dna solution under 5 ℃.Add Virahol 0.75ml, solution is mixed gently, at room temperature hatched then 20 minutes.With this dna solution under 5 ℃ with 14, centrifugal 15 minutes of 000rpm removes supernatant liquor, with 70% washing with alcohol DNA deposit seeds, drying, resuspension in the TE damping fluid again.
7.1.4. Isolated plasmid dna from intestinal bacteria
(Bacto-peptone-10g, Bacto-yeast extract-5g and NaCl-10g are at 1 liter of dH in 5ml Luria-Bertani (LB) substratum with the intestinal bacteria bacterium colony of single conversion 2Among the O, pH7.0 121 ℃ of following autoclavings 25 minutes, and replenishes with 100 μ g/ml ampicillins) hatch.With the culture incubated overnight, 1 μ m aliquots containig is placed the miniature centrifuge tube of 1.5ml, culture sample is loaded onto AutoGen 540 TMAutomatically in the separate nucleic acid device,, use Cycle 3 (instrument software) isolated plasmid dna according to operational manual.
7.1.5. The preparation of deinsectization streptomycete protoplastis and conversion
The single bacterium colony of deinsectization streptomycete is separated in the YPD-6 of 1/2 concentration.Mycelium is used for being inoculated in the 10ml TSB substratum of 25mm * 150mm test tube, and under 28 ℃ with 300rpm shaking culture 48 hours.With 1ml mycelium inoculation 50ml YEME substratum.Every liter of YEME substratum contains: Difco Yeast Extract-3g; Difco Bacto--5g; DifcoMalt Extract-3g; Sucrose-300g.Through after 25 minutes, adding following material: 2.5M MgCl at 121 ℃ of following autoclavings 26H 2O (respectively 121 ℃ of following autoclavings 25 minutes)-2ml; And glycine (20%) (filtration sterilization)-25ml.
Mycelium was cultivated 48-72 hour down at 30 ℃, then in 50ml centrifuge tube (Falcon) 3, centrifugal 20 minutes results under the 000rpm.Remove supernatant liquor, mycelium is resuspension in the P damping fluid, and the P damping fluid contains: sucrose-205g; K 2SO 4-0.25g; MgCl 26H 2O-2.02g; H 2O-600ml; K 2PO 4(0.5%)-10ml; Trace element solution-20ml; CaCl 22H 2O (3.68%)-100ml; And the MES damping fluid (1.0M, pH6.5)-(the * trace element solution contains for every liter 10ml: ZnCl 2-40mg; FeCl 36H 2O-200mg; CuCl 22H 2O-10mg; MnCl 24H 2O-10mg; Na 2B 4O 710H 2O-10mg; (NH 4) 6Mo 7O 244H 2O-10mg).PH transfers to 6.5, and final volume transfers to 1 liter, with 0.45 micron sieve heat filtering substratum.
Mycelium is with 3, and 000rpm obtained granular precipitation in centrifugal 20 minutes, removes supernatant liquor, with mycelium resuspension in containing the 20ml P damping fluid of 2mg/ml N,O-Diacetylmuramidase.Mycelium vibrates down at 35 ℃ and hatched 15 minutes, examines under a microscope to determine to form the scope of protoplastis.When protoplastis form to finish, with it 8, under the 000rpm centrifugal 10 minutes.Remove supernatant liquor, with protoplastis resuspension in 10ml P damping fluid.8, centrifugal 10 minutes of 000rpm removes supernatant liquor, with its resuspension in 2ml P damping fluid, nearly 1 * 10 with protoplastis 9Individual protoplastis is distributed in the 2.0ml low temperature bottle (Nalgene).
Be equipped with 1 * 10 9The bottle of individual protoplastis is 8, and under the 000rpm centrifugal 10 minutes, remove supernatant liquor, protoplastis is resuspension in 0.1ml P damping fluid.The DNA that 2～5 μ g are transformed joins in the protoplastis, then adds 0.5ml work T damping fluid.T buffering base contains: PEG-1000 (Sigma)-25g; Sucrose-2.5g; H 2O-83ml..With pH regulator to 8.8 (after the filter-sterilized), T buffering base is filter sterilization and preserves down at 40 ℃ with the NaOH of 1N.Work T damping fluid (made the same day and use) is T buffering base-8.3ml; K 2PO 4(4mM)-1.0ml; CaCl 22H 2O (5M)-0.2ml; And TES (1M, pH8)-0.5ml..Each component of this work T damping fluid all is the difference filter sterilization.
In 20 seconds, the T damping fluid is joined protoplastis, adds 1.0ml P damping fluid simultaneously, then with protoplastis 8, centrifugal 10 minutes of 000rpm.Remove after the supernatant liquor protoplastis resuspension in 0.1ml P damping fluid.Then protoplastis is cultivated at the RM14 culture medium flat plate, this substratum contains: sucrose-205g; K 2SO 4-0.25g; MgCl 26H 2O-10.12g; Glucose-10g; Difco Casamino Acids-O.1g; Difco Yeast Extract-5g; Difco Oatmeal agar-3g; Difco Bacto agar-22g; DH 2O-800ml.Solution was 121 ℃ of following autoclavings 25 minutes.Through sterilization, add following sterile component: K 2PO 4(0.5%)-10ml; CaCl 22H 2O (5M)-5ml; L-proline(Pro) (20%)-15ml; The MES damping fluid (1.0M, pH6.5)-10ml; Trace elements (the same)-2ml; Cycloheximide stoste (25mg/ml)-40ml; And 1N NaOH-2ml.On each flat board, these flat boards are all wanted dry 24 hours before use with 25ml RM14 substratum five equilibrium.
Protoplastis is in humidity 95%, hatches 20-24 hour under 30 ℃ of the temperature.In order to select thiostrepton resistance transformant, will contain the streptothricial 1ml repetition of 125 μ g/ml sulphur damping fluid and be tiled in equably on the RM14 regeneration plate, every 100ml repeats damping fluid and contains: sucrose-10.3g; Trace element solution (with last identical)-0.2ml; And MES (1M, pH6.5)-1ml.Protoplastis is in humidity 95%, hatches under 30 ℃ of the temperature to occur up to thiostrepton resistance (ThiO ') bacterium colony in 7-14 days.
7.1.6. The conversion of the purple protoplastis of streptomycete
In some cases, purple protoplastis (S.lividans) TK64 of streptomycete (by JohnInnes institute, Norwich, U.K provides) be used to transform.The method of S.lividans growth, primary materialization and conversion and component are people such as Hopwood, 1985, the genetic manipulation of streptomycete (Genetic Manipulation of Streptomyces, A Laboratory Manual, JohnInnes Foundation, Norwich, the existing description among the U.K., and carry out as described therein.Described in the separation such as above-mentioned 7.1.3 of plasmid DNA from the S.lividans transformant.
7.1.7. The fermentation analysis of deinsectization streptomycete strain
The mycelium of deinsectization streptomycete is after cultivating 4-7 days on the YPD-6 of 1/2 concentration, be inoculated in 1 * 6 inch the test tube, wherein contain substratum and two 5mm glass beads that 8ml is prefabricated into, the substratum that is prefabricated into contains: Zulkovsky starch (perhaps lower boiling starch or KOSO, JapanCom Starch Co., Nagoya)-20g/L; Pharmamedia-15g/L; ArdaminepH-5g/L (Champlain Ind., Clifton, NJ); CaCO 3-2g/L; 2x bcfa (" bcfa ' " refers to branched chain fatty acid) contains a ultimate density in substratum be 50ppm 2-(+/-)-methylbutyric, 60ppm isopropylformic acid, and 20ppm isovaleric acid.PH is transferred to 7.2, and substratum was 121 ℃ of following autoclavings 25 minutes.
Test tube with 17 ° of angles under 29 ℃ with 215rpm shaking culture 3 days.To be inoculated into the aliquots containig of 2-ml seed culture in the 300ml Erlenmeyer flask, and wherein contain the 25ml growth medium, this substratum contains: starch (perhaps lower boiling starch or KOSO)-160g/L; Nutrisoy (Archer Daniels Midland, Decatur, IL) 10g/L; Ardamine pH-10g/L; K 2HPO 4-2g/L; MgSO 44H 2O-2g/L; FeSO 47H 2O-0.02g/L; MnCl 2-0.002g/L; ZnSO 47H 2O-0.002g/L; CaCO 3-14g/L 2x bcfa (the same); And hexahydrobenzoic acid (CHC) (under pH7.0, making 20% solution)-800ppm.PH transfers to 6.9, and substratum was 121 ℃ of following autoclavings 25 minutes.
After the inoculation, with flask under 29 ℃ with 200rpm shaking culture 12 days.Through cultivating, from flask, extract the sample of 2ml, with the dilution of 8ml methyl alcohol, mix, mixture is centrifugal 10 minutes shards under 1,250 * g.Pass through analytically clear liquid of HPLC with Beckman Ultrasphere ODS pillar (25cm * 4.6mm ID), flow velocity is 0.75 ml/min, and by the absorbance detection under 240nm.Moving phase is 86/8.9/5.1 methanol/acetonitrile.
7.1.8. The separation of deinsectization streptomycete PKS gene
(ATCC 31272, and SC-2) cosmid library of chromosomal DNA is used ketone synthetic enzyme (KS) probe of being made by many spores of redness bacterium polyketide synthases (PKS) gene fragment to be prepared and hybridized for deinsectization streptomycete.Being described in detail in following document and can finding of relevant cosmid library preparation; People such as Sambrook, 1989, the same.Being described in detail in following document and can finding of relevant streptomycete chromosome DNA library preparation; People such as Hopwood, 1985, the same.Contain ketone synthetic enzyme-hybridization zone the clay bacterium colony can by with the Ndel/Eco47III fragment hybridization identification of a 2.7Kb from pEX26 (by Dr.P.Leadlay, Cambridge, UK provides).Use Ndel and Eco47III, nearly 5ng pEX26 is digested.Reaction mixture is stated from 0.8%SeaPlaque GTG sepharose, and (FMC BioProducts, Rockland is on the ME.From gel, excise the 2.7KbNdel/Eco47III fragment behind the process electrophoresis, and use the GELase of Fast Protocol TM(EpicentreTechnologies) from gel, reclaim DNA.2.7Kb the Ndel/Eco47III fragment with [α- 32P] dCTP (Deoxyribose cytidine 5 '-triphosphate, four (triethylamine) salt, [alpha- 32P]-) ((BRL Life Technologies, Inc., Gaithersburg MD) carry out mark MA) to use BRL Nick translation system according to supplier's guidance for NEN-Dupont, Boston.In the 0.05ml volume, typically react.After adding 5 μ l stop buffers, instruct, with the dna molecular of mark G-25 Sephadex Quick Spin according to manufacturer TMColumn (Boehringer Mannheim) separates from the nucleic acid molecule of not integrating as yet.
About 1,800 clay bacterium colony screens by colony hybridization.Identify 10 bacterium colonies of hybridizing with red many spores bacterium KS probe brute force.The intestinal bacteria bacterium colony that includes cosmid DNA is grown in the LB liquid nutrient medium, uses Cycle 3 (instrument software) at AutoGen 540 according to operation instructions TMAutomatically from each culture, separate cosmid DNA in the separate nucleic acid device.Restriction endonuclease map and southern blotting technique hybridization analysis have disclosed in five kinds of bacterium colonies and have contained the eclipsed chromosomal region.Five kinds of clays (also be pSE65, pSE66, pSE67, pSE68, deinsectization streptomycete genome BamHl restriction enzyme mapping pSE69) makes up (Fig. 4) by analyzing eclipsed clay and hybrid method.
7.1.9. Regulation and control avermectin B2: the DNA evaluation of B1 ratio and the evaluation of aveC ORF
Following method is used for testing the subclone fragment regulation and control AveC mutant strain avermectin B2 that comes from pSE66 clay clone: the ability of B1 ratio.With Sacl and BamHl digestion pSE66 (5 μ g).Reaction mixture is stated from 0.8%Seaplaque TMGTG sepharose (FMC BioProducts), 2.9Kb Sacl/BamHl fragment is cut the gel behind electrophoresis, and uses the GELase of Fast Protocol TM(Epicentre Technologies) reclaims DNA from gel.The shuttle vectors pWHM3 of nearly 5 μ g (people such as Vara, 1989, J.Bacteriol.171:58 72-5881) is digested by Sacl and BamHI.The pWHM3 that has the 2.9Kb of 0.5 μ g to insert son and 0.5 μ g digestion is mixed together, and (Beverly is an overnight incubation under the 20 μ l conditions at 15 ℃, cumulative volume MA) for New England Biolabs, Inc. with the ligase enzyme of 1 unit according to manufacturer's explanation.Through hatching, the connection mixture of 5 μ l was hatched under 70 ℃ 10 minutes, was cooled to room temperature, was used for the E.coli DH5 α cell (BRL) of transformed competence colibacillus according to operational manual.Plasmid DNA is separated from ampicillin resistance transformant, can confirm that by restricted enzyme cutting analysis 2.9Kb Sacl/BamHI inserts the existence of son.This plasmid is named as pSE119.
As above the 7.1.5 part is described, and the protoplastis of deinsectization streptomycete bacterial strain 1100-SC38 (the inner bacterial strain of Pfizer) is prepared and transforms with pSE119.Strain 1100-SC38 is a muton, when replenishing with hexahydrobenzoic acid, compares with avermectin cyclohexyl-B1 form, and it produces obviously many avermectin cyclohexyl-B2 forms (ratio of B2: B1 approximately is 30: 1).The pSE119 that is used to transform the deinsectization streptomycete protoplastis (derives from Dr.B.J.Bachmann from E.coli strain GM2163, Curator, E.coli Genetic Stock Center, YaleUniversity), separate among E.coli strain DM1 (BRL) or the S.lividans strain TK64.The thiostrepton resistance transformant of isolated strains 1100-SC38 is also analyzed leavened prod by HPLC and is analyzed.Contain the avermectin cyclohexyl B2 that the transformant generation ratio of the deinsectization streptomycete strain 1100-SC38 of pSE119 changes: cyclohexyl-B1, this ratio approximately is 3.7: 1 (table 2).
Determined that pSE119 can regulate and control the avermectin B2 in the AveC muton: after the ratio of B1, measure the dna sequence dna of insertion.According to operational manual, (CA), the pSE119 of nearly 10 μ g is separated for Qiagen, Valencia, and (Perkin Elmer, Foster City CA) checks order to use ABI 373A automated DNA sequenator then to use the plasmid DNA separating kit.With genetics computer set program (GCG, Madison, WI) combination and editor's sequencing data.Dna sequence dna and aveC ORF list in Fig. 1 (SEQ ID NO:1).
A kind of new plasmid of following structure, called after pSE118.Digest the pSE66 of about 5 μ g with SphI and BamHI.Reaction mixture is loaded on the 0.8%SeaPlaque GTG sepharose (FMCBioProducts), and the SphI/BamHI fragment of 2.8Kb is excised from gel through electrophoresis, uses the GELase of Fast Protocol TM(Epicentre Technologies) reclaims DNA from gel.The shuttle vectors pWHM3 of nearly 5 μ g is digested by SphI and BamHI.There are 2.8Kb insertion of 0.5 μ g and the pWHM3 of 0.5 μ g digestion to be mixed together, and be according to ligase enzyme (the New England Biolabs of manufacturer's explanation under the 20 μ l conditions with 1 unit at 15 ℃, cumulative volume, Inc., Beverly, MA) overnight incubation therewith.Through hatching, the connection mixture of 5 μ l was hatched under 70 ℃ 10 minutes, was cooled to room temperature, was used to transform the E.coliDH5 α cell of being felt attitude according to operational manual.Plasmid DNA is separated from ampicillin resistance transformant, can confirm that by restricted enzyme cutting analysis 2.8Kb SphI/BamHI inserts the existence of son.This plasmid is named as pSE118.Nearly 838 Nucleotide overlapping (table 4) among the sub-DNA of insertion in pSE118 and pSE119.
The protoplastis of deinsectization streptomycete strain 1100-SC38 as above transforms with pSE118.The thiostrepton resistance transformant of isolated strains 1100-SC38 is also analyzed leavened prod by HPLC and is analyzed.The transformant of deinsectization streptomycete strain 1100-SC38 that contains pSE118 with respect to strain 1100-SC38 at avermectin cyclohexyl B2: change on the ratio of cyclohexyl-B1 by (table 2).
7.1.10. Pcr amplification from the aveC gene of deinsectization streptomycete chromosomal DNA
One contain aveC ORF-the 1.2Kb fragment separates from the deinsectization streptomycete chromosomal DNA by the pcr amplification method, the primer of its use according to from the aveC nucleotide sequence that obtained design.The PCR primer is by Genosys Biotechnologies, and Inc. (Texas) provides.Primer is to the right: 5 '-TCACGAAACCGGACACAC-3 (SEQ ID NO:6); Primer is left: 5 '-CATGATCGCTGAACCGAG-3 ' (SEQ ID NO:7).In by the damping fluid that the manufacturer provided, use Deep Vent TMPolysaccharase (New England-Biolabs) carries out the PCR reaction, and has 300 μ M dNTP, 10% glycerine, and every kind of primer of 200pmol, 0.1 μ g template and 2.5 unit enzymes, final volume are 100 μ l, use Perkin-Elmer Cetus heat exchanger.First round-robin thermal map is 95 ℃ 5 minutes (denaturing steps), 60 ℃ 2 minutes (annealing steps), 72 ℃ following 2 minutes (extension step).24 circulations subsequently have a similar Thermogram, but the time of denaturing step foreshortens to 45 seconds kinds and annealing time foreshortens to 1 minute.
The PCR product behind the electrophoresis, detects single DNA band of one-1.2Kb in 1% sepharose.This DNA of purifying from gel, and be 1: 10 mole carrier a ratio with the blunt PCR-inactive base (Invitrogen) of 25ng linearity according to operational manual: insert son and connect.To connect mixture according to operational manual and be used to transform Shot TMCompetent E.coil cell (Invitrogen).Plasmid DNA is separated from the amicillin resistance transformant, should～existence that 1.2Kb inserts son can confirm by restricted enzyme cutting analysis.This plasmid is named as pSE179.
From the sub-DNA of the insertion of pSE179 by digesting separation with BamHI/XbaI, separate with electrophoretic method, purifying from gel, and connecting (also being digested by BamHI/XbaI) with shuttle vectors pWHM3, it is that 1: 5 mole carrier carries out with inserting in the total DNA concentration of 1 μ g that son exists with ratio at one.Connect the E.coilDH5 α cell that mixture is used for transformed competence colibacillus according to operational manual.Plasmid DNA is separated from the amicillin resistance transformant, should～existence that 1.2Kb inserts son can confirm by restricted enzyme cutting analysis.This plasmid (being named as pSE186 (Fig. 2, ATCC 209604)) is transformed among the E.coil DM1, and plasmid DNA is separated from the amicillin resistance transformant.
7.2. The result
Identify 2.9Kb SacI/BamHI endonuclease bamhi, when it is converted among the deinsectization streptomycete strain 1100-SC38, changed B2 significantly: the rate ratio of B1 avermectin from pSE119.Generally speaking, deinsectization streptomycete strain 1100-SC38 B2: the ratio of B1 is about 30: 1, but when transforming with the carrier that contains 2.9Kb SacI/BamHI endonuclease bamhi, the ratio of avermectin B2: B1 is reduced to about 3.7: 1.The transformant culture is carried out the existence that the secondary fermentation analysis has confirmed transfering DNA.
2.9Kb pSE119 fragment is checked order, and identified the ORF (Fig. 1) (SEQ ID NO:1) of about 0.9Kb, it comprises a PstI/SphI fragment, and it only produces B2 product (people such as Ikeda, 1995, the same) when undergoing mutation in other place in the past.This ORF or its corresponding inductive polypeptide, (GenEMBL SWISS-PROT) compares, and does not show that itself and known dna or protein sequence have any homology with the given data storehouse.
The fermentation analysis of the deinsectization streptomycete strain 1100-SC38 that table 2 expression transforms with different plasmids
Table 2
Deinsectization streptomycete strain (conversion plasmid) The transformant Test No. Average B2: B1 ratio
1100-SC38 (nothing) 9 ?30.66
1100-SC38(pWHM3) 21 ?31.3
1100-SC38(pSE119) 12 ?3.7
1100-SC38(pSE118) 12 ?30.4
1100-SC38(pSE185) 14 ?27.9
8. embodiment: The structure of deinsectization streptomycete Avec mutant strain
Present embodiment has been described and has been utilized said components and method to make up several different deinsectization streptomycete AveC mutant strains.Can be with the technology that mutant strain is introduced in the streptomyces gene referring to Kieser and Hopwood, 1991, the general description of Meth.Enzym.204:430-458.More detailed explanation can be referring to people such as Anzai, and 1988, J.Antibiot XLI (2): people such as 226-233 and Stutzman-Engwall, 1992, J.Bacteriol.174 (1): 144-154.These bibliographys are all quoted in full at this.
8.1. The inactivation of deinsectization streptomycete AveC gene
The AveC mutant strain that contains the AveC gene of inactivation can make up by several method as described below.
In first method, the ermE gene that SphI/PstI fragment at the 640bp of pSE119 (the as above described plasmid of 7.1.9) aveC gene inside is come from Sacc.erythraea replaces (for the erythromycin resistance).Digest by restriction enzyme digestion with Bg/II and EcoRI, from pIJ4026 (from John Innes Institute, Norwich, U.K.; Also referring to people such as Bibb, 1985, Gene 41:357-368) separate the ermE gene in, carry out electrophoresis then, and from gel purifying.This about 1.7Kb fragment is connected to pGEM7Zf (Promega), and it digests with BamHI and EcoRI, and according to operational manual, will connect mixture and be transformed in the competent E.coli DH5 α cell.Plasmid DNA is separated from the amicillin resistance transformant, and the existence that this about 1.7Kb inserts son can confirm by restricted enzyme cutting analysis.This plasmid is named as pSE27.
PSE118 (as above 7.1.9 partly describes) digests with SphI and BamHI, and digest is carried out electrophoresis, and should insert son purifying from gel by about 2.8Kb SphI/BamHI.PSE119 digests with PstI and EcoRI, carries out electrophoresis then, and should insert son purifying from gel by about 1.5Kb PstI/EcoRI.Shuttle vectors pWHM3 is digested with BamHI and EcoRI.PSE27 digests with PstI and SphI.Carry out electrophoresis then, and PstI/SphI that will about 1.7Kb inserts son purifying from gel.All these four fragments (that is: about 2.8Kb, about 1.5Kb, about 7.2Kb, about 1.7Kb) link together with four kinds of forms.According to operational manual, will connect mixture and be transformed in the competent E.coli DH5 α cell.Isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis.This plasmid is named as pSE180 (Fig. 3; ATCC209605).
PSE180 is transformed into S.lividans TK64 cell, identifies the bacterium colony that has transformed by anti-thiostrepton and erythromycin microbiotic.From S.Lividans, separate pSE180, and be used to transform the protoplastis of deinsectization streptomycete.Identify four kinds of thiostrepton resistance deinsectization streptomycete transformant, and prepare protoplastis and it is carried out bed board in the RM14 of non-selective condition substratum.Behind the protoplast regeneration, screening has the erythromycin resistance and does not have single bacterium colony of thiostrepton resistance, has shown the chromosomal integration of inactivation aveC gene and the disappearance of free replicon.Identify Erm rThio sTransformant, and with its called after SE180-11 bacterial strain.Whole chromosomal DNA is separated from the SE180-11 strain, and use Restriction Enzyme BamHI, HindIII, PstI or SphI digest, and in 0.8% sepharose, carry out electrophoretic separation, be transferred on the nylon membrane then, and hybridize with the ermE probe.These analyze demonstration, and the chromosomal integration of ermE resistant gene and the segmental disappearance of the 640bpPstI/SphI that follows are to be taken place by the process of a dual exchange.The HPLC of SE180-11 strain leavened prod analyzes and shows that general avermectin no longer produces (Fig. 5 A).
In the second method that makes the aveC gene inactivation, 1.7Kb ermE gene is removed from the karyomit(e) of deinsectization streptomycete SE180-11 strain, stays next 640bp PstI/SphI disappearance in the aveC gene.Following structure gene replaces plasmid: partly digest pSE180 with XbaI, and will about 11.4Kb fragment purifying from gel.This about 11.4Kb band lacks 1.7Kb ermE resistant gene.E.coli DH5 α cell is advanced in this DNA connection and conversion then.Isolated plasmid dna from the amicillin resistance transformant, proofreading and correct the existence of inserting son can confirm by restricted enzyme cutting analysis.This plasmid (being named as pSE184) is transformed among the E.coli DM1, isolated plasmid dna from the amicillin resistance transformant.This plasmid is used to transform the protoplastis of deinsectization streptomycete SE180-11 strain.From the thiostrepton resistance transformant of SE180-11 strain, prepare protoplastis, and on RM14, carry out bed board as single bacterium colony.Behind the protoplast regeneration, filter out single bacterium colony of no erythromycin resistance and thiostrepton resistance, shown the chromosomal integration of inactivation aveC gene and contained the disappearance of the free replicon of ermE gene.Identify Erm rThio sAnd called after SE184-1-13.The general avermectin of demonstration is analyzed in the fermentation of SE184-1-13 no longer produced, SE184-1-13 has the fermenting characteristic identical with SE180-11.
Make in the method for aveC gene inactivation at the third, use PCR method a frameshit to be introduced on the karyomit(e) aveC gene, therefore produce a BspEl site at two G of adding behind the C of nucleotide position 471.The existence of the EspEl position of processing is useful for detecting the gene substitution process.Design PCR primer is to introduce phase shift mutation in the aveC gene, this primer is provided by GenosysBiotechnologies company.Primer is to the right: 5 '-GGTTCCGGATGCCGTTCTCG-3 ' (SEQ ID NO:8) left primer be: 5 '-AACTCCGGTCGACTCCCCTTC-3 ' (SEQ IDNO:9).The condition of carrying out PCR as above 7.1.10 part is described.With the PCR product of Sphl digestion 666bp, provide the fragment of two 278 bp and 388 bp respectively.Purifying 388bp fragment from gel.
The following structure of gene-altered plasmid: with EcoRI and BamHI digestion shuttle vectors pWHM3.With BamHI and SphI digestion pSE119, carry out electrophoresis then, from gel, separate the fragment of about 840bp.With EcoRI and XmmI digestion pSE119, carry out electrophoretic separation then, and from gel the fragment of the about 1.7Kb of purifying.Four kinds of fragments (also promptly :～7.2Kb ,～840bp ,～1.7Kb and 388bp) link together with four kinds of different modes.Connecting mixture is transformed in the into competent E.coliDH5 α cell.Isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis and dna sequence analysis.This plasmid (called after pSE185) transforms and enters E.coli DM1 cell, isolated plasmid dna from the amicillin resistance transformant.This plasmid is used to transform the protoplastis of deinsectization streptomycete strain 1100-SC38.The thiostrepton resistance transformant of isolated strains 1100-SC38, and analyze leavened prod by HPLC and analyze.When pSE185 is transformed among the deinsectization streptomycete strain 1100-SC38, obviously do not change the ratio (table 2) of avermectin B2: B1.
PSE185 is used for transforming the deinsectization streptomycete protoplastis to produce a phase shift mutation at karyomit(e) aveC gene.Prepare protoplastis from thiostrepton resistance transformant, and on the RM14 substratum as single bacterium colony bed board.Behind the protoplast regeneration, screen single bacterium colony of no thiostrepton resistance.By PCR separate and screening from the existence that is integrated into the phase shift mutation in the karyomit(e) of the chromosomal DNA of the bacterium colony of thiostrepton sensitivity.This PCR primer is classified basic design as with the aveC nucleotides sequence, is provided by Genosys Biotechnologies (Texas) company.PCR primer to the right is: 5 '-GCAAGGATACGGGGACTAC-3 ' (SEQ ID NO:10), PCR primer left is: 5 '-GAACCGACCGCCTGATAC-3 ' (SEQ ID NO:11), as above the 7.1.10 part is described to carry out the condition of PCR.The PCR product that is obtained is 543bp, when digesting with BspE1, can detect three fragment 368bp, and 96bp, and 79bp have shown the chromosomal integration of inactivation aveC gene and the disappearance of free replicon.
The general avermectin of deinsectization streptomycete mutant strain fermentation analysis revealed that contains phase shift mutation on the aveC gene no longer produces, and these mutant strains have the HPLC fermenting characteristic identical with strain SE180-11 and SE184-1-13.Thio sTransformant is identified and called after SE185-5a.
In addition, produce sudden change on the aveC gene, make nucleotide position 520 become A by G, consequently the codon of coding colors propylhomoserin (W) is changed into a terminator codon on position 116.There is the deinsectization streptomycete strain of this sudden change not produce general avermectin and has and strain SE180-11, SE184-1-13, and the identical fermenting characteristic of SE185-5a.
In addition, the sudden change that produces at the aveC gene has changed two positions: (i) nucleotide position 970 becomes A by G, make amino acid position 256 become aspartic acid (D) from glycine (G), and (ii) nucleotide position 996 becomes C by T, makes amino acid position 275 become Histidine (H) from tyrosine (Y).Deinsectization streptomycete strain with these sudden changes (G256D/Y275H) does not produce general avermectin and has and strain SE180-11, SE184-1-13, and the identical fermenting characteristic of SE185-5a.
Deinsectization streptomycete aveC inactivation mutant strain SE180-11, SE184-1-13, SE185-5a, and other strain mentioned herein provide screening implement to be used to assess the influence of other sudden change of aveC gene.The pSE186 that contains the wild-type copy of aveC gene is transformed in the E.coil DM1 cell, and plasmid DNA is separated from the amicillin resistance transformant.This pSE186 DNA is used to transform the SE180-11 strain of deinsectization streptomycete.The thiostrepton resistance transformant of separating the SE180-11 strain is measured the existence of erythromycin resistance, analyzes leavened prod by HPLC and analyzes Thio rErm rTransformant.The existence of the functional aveC gene of antiposition just can return to normal disinsection mychel yield SE180-11 strain (Fig. 5 B).
8.2 Change B2: the mutation analysis of the AveC gene of B1 ratio
As mentioned above, the deinsectization streptomycete bacterial strain SE180-11 that contains the aveC gene of inactivation replenishes by transforming with a plasmid (pSE186) that contains functional aveC gene.The SE180-11 strain also can be used as other sudden change that host strain is identified the aveC gene, and is as described below.
From bacterial strain 1100-SC38, separate chromosomal DNA, and carry out the template of pcr amplification as the aveC gene.Separate 1.2Kb ORF by pcr amplification, the employed primer that increases is classified the basis as with the aveC nucleotides sequence and is designed.Primer to the right is SEQ ID NO:6, and primer left is SEQ IDNO:7 (described referring to above-mentioned 7.1.10 part).PCR and subclone condition are as described in the 7.1.10 part.Become the aveC transgenation of T 1.2Kb the dna sequence analysis of ORF has shown at nucleotide position 337 by C, the amino acid at 55 places becomes phenylalanine (F) by Serine (S) in the position.The aveC gene subclone that contains the S55F sudden change is gone into pWHM3 to produce a plasmid, and it is named as pSE187, and is used to transform the protoplastis of deinsectization streptomycete bacterial strain SE180-11.The thiostrepton resistance transformant of separating the SE180-11 strain to determine existing of erythromycin resistance, is analyzed leavened prod by HPLC and is analyzed Thio rErm rTransformant.The existence of the aveC gene that changes on the coded amino acid residue 55 (S55F) can return to normal disinsection mychel yield SE180-11 strain (Fig. 5 C); Yet, cyclohexyl B2: the ratio of cyclohexyl B2 approximately is 26: 1, compare with the SE180-11 strain that transforms with pSE186, the ratio of its B2: B1 approximately is 1.6: 1 (table 3), has shown that single sudden change (S55F) regulated and control the output with respect to the cyclohexyl B2 of cyclohexyl B1.
Identify another sudden change in the aveC gene, make nucleotide position 862 become A by G, the amino acid at 230 places, position becomes aspartic acid (D) by glycine (G).The avermectin B2 that contains the deinsectization streptomycete strain generation of this sudden change (G230D): the ratio of B1 approximately is 30: 1.
8.3. Reduce B2: the sudden change of B1 ratio
Can reduce several sudden changes of cyclohexyl-B2 by following structure to cyclohexyl-B1 ratio.
Identify the sudden change of aveC gene, make nucleotide position 588 become A by G, the amino acid at 139 places, position becomes Threonine (T) by L-Ala (A).The aveC gene that contains A139T sudden change is gone among the pWHM3 producing a plasmid by subclone, its called after pSE188, and it is used to transform the protoplastis of deinsectization streptomycete SE180-11 strain.The thiostrepton resistance transformant of separating the SE180-11 strain is measured the existence of erythromycin resistant strain, analyzes leavened prod by HPLC and analyzes Thio rErm rTransformant.The existence of the aveC gene of the sudden change that coded amino acid residue 139 (A139T) changes can return to disinsection mychel yield SE180-11 strain (table 5D); Yet the ratio of B2: B1 is 0.94: 1 approximately, and this shows that this sudden change has reduced the output of cyclohexyl B2 with respect to cyclohexyl B1.This result is not expection, because the result of result who announces and said mutation has only proved the inactivation of aveC gene or increased the output (see Table 3) of avermectin B2 form with respect to the B1 form.
Because A139T sudden change has changed the ratio of B2: B1 with suitable more B1 direction, therefore make up the Threonine of coded amino acid position 138 but not the sudden change of Serine.Like this, with EcoRI digestion pSE186 and be cloned among the pGEM3Zf (Promega) that has been digested by EcoRI.This plasmid that is named as psE186a digests with ApaI and KpnI, DNA isolation fragment in sepharose, and two fragment～3.8Kb of purifying reach～0.4Kb from gel.From pSE186～dna fragmentation that 1.2Kb inserts is used as a pcr template to bring out the change that single base takes place on nucleotide position 585.The PCR design of primers is become can introduce a sudden change at nucleotide position 585, and this primer is to be provided by GenosysBiotechnologies company (Texas).PCR primer to the right is: 5 '-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3 ' (SEQ ID NO:12); PCR primer left is: 5 '-GGAACCGACCGCCTGATACA-3 ' (SEQ ID NO:13).Use advanced GC genome PCR test kit (Clonetech Laboratories, Palo Alto, CA) in the damping fluid that provides by manufacturer, there are 200 μ M dNTPs, every kind of primer of 200pmol, the 50ng template DNA, 1.0M under the KlenTaq polymerase mixture of GC-Melt and 1 unit, final volume is 50 μ l, carries out the PCR reaction.The first round-robin thermal profile be 94 ℃ 1 minute, next 25 circulations all are to carry out in 2 minutes with 94 ℃ of 30 second and 68 ℃, and 68 ℃ of circulations 3 minutes.The PCR product of 295bp is digested to discharge the fragment of a 254bp with ApaI and KpnI, and it is differentiated and purifying from gel by electrophoresis.All three kinds of fragments (～3.8Kb, -0.4Kb and 254bp) link together with three kinds of modes.Be transformed in the complete E.coil DH5 α cell connecting mixture.Plasmid DNA is separated from the amicillin resistance transformant, proofreaies and correct the existence of inserting son and can confirm by restricted enzyme cutting analysis.This plasmid is named as pSE198.
PSE198 digests with EcoRI, and the clone enters among the pWHM3 with EcoRI digestion, and its conversion is entered in the E.coil DH5 α cell.Isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis and dna sequence analysis.This plasmid DNA transforms and enters E.coil DM1, and isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis.This plasmid is named as pSE199, can be used to transform deinsectization streptomycete strain SE180-11 protoplastis.The thiostrepton resistance transformant of isolated strains SE180-11 is determined the existence of erythromycin resistance, analyzes Thio by analyzing leavened prod with the HPLC method rErm rTransformant.The existence that is coded in amino-acid residue 138 (S138T) sudden change aveC gene that changes can return to strain SE180-11 with normal disinsection mychel yield; Yet the ratio of B2: B1 is 0.88: 1, has shown that this sudden change has reduced the quantity (see Table-3) of cyclohexyl-B2 with respect to cyclohexyl-B1.The ratio of this B2: B1 even be lower than 0.94: 1 (this is to transform bacterial strain SE180-11 and the A139T that the produces viewed ratio of suddenling change with pSE188, as mentioned above).
Make up another sudden change to introduce a Threonine at amino acid position 138 and 139 places simultaneously.The DNA that comes from about 1.2Kb insertion of pSE186 is used as the template of PCR.The PCR design of primers is become can bring out sudden change at nucleotide position 585 and 588 places, and this primer is provided by Genosys Biotechnologies company (Texas).PCR primer to the right is: 5 '-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3 ' (SEQ ID NO:14); PCR primer left is: 5 '-GGAACATCACGGCATTCACC-3 ' (SEQ ID NO:15).Use the above-mentioned condition in this part to carry out the PCR reaction.The PCR product that digests 449bp with Apal and Kpnl is to discharge the fragment of 254bp, and it is differentiated and purifying from gel by electrophoresis.PSE186a is digested with ApaI and KpnI,, and two fragments of 3.8Kb and 0.4Kb are purified from gel in agarose gel electrophoresis DNA isolation fragment.All these three fragments (～3.8Kb ,～0.4Kb and 254bp) connect in three kinds of modes, connect mixture and are entered in the active E.coliDH5 α cell by conversion.Isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis.This plasmid is named as pSE230.
Digest pSE230 with EcoRI, the clone advances in the pWHM3 plasmid that is digested by EeoRI, and transforms and enter in the E.coli DH5 α cell.Isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis and dna sequence analysis.This plasmid DNA is entered in the E.coli DM1 cell by conversion, and isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the existence of inserting son by restricted enzyme cutting analysis.This plasmid, it is named as pSE231, is used to transform the protoplastis of deinsectization streptomycete strain SE180-11.Separate the thiostrepton resistance transformant of SE180-11, determine the existence of erythromycin resistant strain, analyze Thio by fermentation then rErm rThe existence of the aveC gene of the dual sudden change of coding S138T/A139T can make normal disinsection mychel yield return to the SE180-11 strain; Yet the ratio of B2: B1 is 0.84: 1, has shown with the reduction that is provided with pSE188 or pSE199 conversion SE180-11 strain to compare, and this sudden change has further reduced the output (see Table 3) of cyclohexyl-B2 with respect to cyclohexyl B1.
Table 3
Deinsectization streptomycete strain (conversion plasmid) The transformant Test No. The B2 relative concentration The B1 relative concentration Average B2: B1 ratio
SE180-11 (nothing) 30 ?0 ?0 ?0
?SE180-11(pWHM3) 30 ?0 ?0 ?0
?SE180-11(pSE186) 26 ?222 ?140 ?1.59
?SE180-11(pSE187) 12 ?283 ?11 ?26.3
?SE180-11(pSE188) 24 ?193 ?206 ?0.94
?SE180-11(pSE199) 18 ?155 ?171 ?0.88
?SE180-11(pSE231) 6 ?259 ?309 ?0.84
These results have proved that for the first time occurring in sudden change specific on the aveC gene has improved the output of commercial needed 1 class avermectin with respect to 2 class avermectins.
9. Embodiment: the structure of 5 ' end deletion mutantion
As top the 5.1st part was explained, there were four different GTG codons at four the bp positions 42,174,177 and 180 places that are listed in to the potential initiation site as the deinsectization streptomycete nucleotides sequence as shown in Fig. 1 (SEQ ID NO:1).AveC ORF (Fig. 1 will be described in this part; SEQ IDNO:1) structure of the multiple disappearance in 5 ' district is to help determining which is used as the initiation site of protein expression in these codons in aveC ORF.
There is the aveC gene fragment of various disappearances from the deinsectization streptomycete chromosomal DNA, to separate at 5 ' end by pcr amplification.The PCR primer is that the basis is designed with the aveC dna sequence dna, and it is provided by GenosysBiotechnologies company.Primer to the right is: 5 '-AACCCATCCGAGCCGCTC-3 ' (SEQ ID NO:16) is (D1F1); 5 '-TCGGCCTGCCAACGAAC-3 ' (SEQ ID NO:17) is (D1F2); 5 '-CCAACGAACGTGTAGTAG-3 ' (SEQ ID NO:18) is (D1F3); And 5 '-TGCAGGCGTACGTGTTCAGC-3 ' (SEQ ID NO:19) is (D2F2).Primer left is: 5 '-CATGATCGCTGAACCGA-3 ' (SEQ ID NO:20); 5 '-CATGATCGCTGAACCGAGGA-3 ' (SEQ ID NO:21); And 5 '-AGGAGTGTGGTGCGTCTGGA-3 ' (SEQ ID NO:22).PCR reaction as above-mentioned the 8.3rd part are carried out.
Separate the PCR product by carry out electrophoresis in 1% sepharose, detected unique DNA band is 1.0Kb or 1.1Kb.Purifying PCR product from gel, and connect with 1: the 10 mole of segmental ratio of carrier-insertion with the linearizing pCR2.1 carrier of 25ng (Invitrogen) according to operational manual.To connect mixture according to operational manual and be used to transform One Shot TMCompetent E.coli cell (Invitrogen).Isolate plasmid DNA from the amicillin resistance transformant, this inserts segmental existence and confirms by restricted enzyme cutting analysis and dna sequence analysis.These plasmids are named as pSE190 (obtaining with primer D1F1), pSE191 (obtaining with primer D1F2), pSE192 (obtaining with primer D1F3) and pSE193 (obtaining with primer D2F2).
The DNA that inserts is digested with BamHI/XbaI respectively, separate by electrophoresis, purifying from gel, and be connected with the shuttle vectors pWHM3 with BamHI/XbaI digestion respectively when to be used in a total DNA concentration be 1 μ g with 1: 5 mole of carrier-insertion fragment ratio.Connect the E.coli DH5 α cell that mixture is used to activity of conversion.From the amicillin resistance transformant, isolate plasmid DNA, insert segmental existence and can confirm by restricted enzyme cutting analysis.These plasmids are named as pSE194 (D1F1), pSE195 (D1F2), pSE196 (D1F3) and pSE197 (D2F2), transformed respectively and entered among the E.Coli strain DM1, isolated plasmid dna from the amicillin resistance transformant confirms to proofread and correct the segmental existence of insertion by restricted enzyme cutting analysis.This DNA is used to transform the protoplastis of deinsectization streptomycete strain SE180-11.The thiostrepton resistance transformant of isolated strains SE180-11 is determined the existence of erythromycin resistance transformant, and it is essential by the aveC expression to determine which GTG site to analyze Thior Errnr transformant by HPLC analysis leavened prod.The result points out that the GTG codon at 42 places in the position can be eliminated, do not influence the expression of aveC, because each lacks the GTG site at 42 places, position pSE194, pSE195 and pSE196, but there are three GTG sites at 174,177 and 180 places in the position, when transforming SE180-11, each can both recover normal disinsection mychel yield.Have only when the SE180-11 strain transforms with the pSE197 that lacks all these four kinds of GTG sites, it just can not recover normal disinsection mychel yield (table 4).
Table 4
Deinsectization streptomycete strain (conversion plasmid) The transformant Test No. The B2 relative concentration The B1 relative concentration Average B2: B1 ratio
SE180-11 (nothing) 6 ?0 ?0 ?0
SE180-11 (pWHM3) 6 ?0 ?0 ?0
SE180-11 (pSE186) 6 ?241 ?152 ?1.58
SE180-11 (pSE194) 6 ?35 ?15 ?2.43
SE180-11 6 ?74 ?38 ?1.97
(pSE195)
SE180-11 (pSE196) 6 ?328 ?208 ?1.58
SE180-11 (pSE197) 12 ?0 ?0 ?0
10. Embodiment: the aveC homologue of clone streptomyces hygroscopicus and product streptomyces griseus
The present invention identify and cloned streptomycete other produce avermectin or than the aveC homologue gene of sweet mycin bacterial classification.For example, the cosmid library of streptomyces hygroscopicus (FERM BP-1901) genomic dna and the 1.2Kb aveC probe hybridization of above-mentioned deinsectization streptomycete.Identify several clay bacterium colonies that very strong hybridization is arranged.Chromosomal DNA is separated from these clays, and identify 4.9Kb KpnI fragment and aveC probe hybridization.Measure the sequence of this DNA, identify the ORF (SEQ ID NO:3) that homology is clearly arranged with the aveC ORF of deinsectization streptomycete.The aminoacid sequence (SEQ ID NO:4) of deriving out from streptomyces hygroscopicus aveC homologue ORF as shown in Figure 6.
In addition, will produce the cosmid library of streptomyces griseus genomic dna and hybridize from the 1.2Kb aveC probe of above-mentioned deinsectization streptomycete.Identify the clay clone of several strong hybridization.From these clays, separate chromosomal DNA, identify a PstI fragment with the 5.4Kb of aveC probe hybridization.This DNA is checked order, and identify the part of O RF that the aveC homologue of homology is clearly arranged with the aveC ORF of deinsectization streptomycete.The partial amino-acid series (SEQ ID NO:5) that derivation is come out as shown in Figure 6.
DNA and amino acid sequence analysis from streptomyces hygroscopicus and the aveC homologue that produces streptomyces griseus show the total each other tangible homology (50% sequence identity is arranged approximately) in these zones or with deinsectization streptomycete aveC ORF and AveC gene product tangible homology (Fig. 6) are arranged also on amino acid levels.
11. Embodiment: after the ermE promotor, use the gene constructed plasmid of aveC
The 1.2Kb aveC ORF of pSE186 by subclone in pSE34, it is a shuttle vectors pWHM3, has 300bp ermE promotor, this promotor is inserted in the KpnI/BamHI site of pWHM3 (referring to people such as Ward as a KpnI/BamHI fragment, 1986, Mol.Gen.Genet 203:468478).With BamHI and HindIII digestion pSE186, differentiate digest by electrophoresis, the 1.2Kb fragment is separated from sepharose and be connected with the pSE34 that digests with BamHI and HindIII.According to operation instructions, this connection mixture is entered in the competent E.coil DH5 α cell by conversion.From the amicillin resistance transformant, isolate plasmid DNA, confirm that by restricted enzyme cutting analysis 1.2Kb inserts segmental existence.This plasmid (being named as pSE189) is transformed to be entered among the E.coli DM1, and isolates plasmid DNA from the amicillin resistance transformant.Transform the protoplastis of deinsectization streptomycete bacterial strain 1100-SC38 with pSE189.The thiostrepton resistance transformant of isolated strains 1100-SC38, and analyze leavened prod by HPLC and analyze.
The ratio (approximately being 3: 1) that contains the cyclohexyl-B2 of the avermectin that the deinsectization streptomycete strain 1100-SC38 transformant of pSE189 produced and cyclohexyl-B1 has changed with respect to the ratio (approximately being 34: 1) of the above-mentioned substance that bacterial strain 1100-SC38 is produced, but compare with the bacterial strain 1100-SC38 that transforms with pSE119, total disinsection mychel yield has increased about 2.4 times (table 5).
PSE189 also can transform in the protoplastis that enters the wild-type deinsectization streptomycete bacterial strain.Separate sulfur Streptothrix resistance polypeptide transformant, and analyze tunning by HPLC and analyze.The avermectin ultimate production of the deinsectization streptomycete wild-type that transforms with pSE189 is compared with the wild-type deinsectization streptomycete strain that transforms with pSE119 and has been increased by 2.2 times (table 5).
Table 5
Deinsectization streptomycete strain (conversion plasmid) The transformant Test No. The B2 relative concentration The B1 relative concentration Relative avermectin ultimate production Average B2: B1 ratio
1100-SC38 6 ?155 ?4.8 ?176 ?33.9
1100-SC38 (pSE119) 9 ?239 ?50.3 ?357 ?4.7
1100-SC38 (pSE189) 16 ?546 ?166 ?849 ?3.3
Wild-type 6 ?59 ?42 ?113 ?1.41
Wild-type (pSE119) 6 ?248 ?151 ?481 ?1.64
Wild-type (pSE189) 5 ?545 ?345 ?1071 ?1.58
12. embodiment: the chimeric plasmid that contains deinsectization streptomycete aveC ORF and streptomyces hygroscopicus aveC homologue sequence
As described below, the heterozygosis plasmid of structure called after pSE350, it contains the streptomyces hygroscopicus aveC homologue of 564bp part, replaces the homologue part (see figure 7) of the 564bp of deinsectization streptomycete aveC ORF.Use BsaAI restriction site and KpnI restriction site to make up pSE350, the BsaAI restriction site is present in (225 of aveC) in two sequences, and the KpnI restriction site is present in (810 of aveC) in the deinsectization streptomycete aveC gene.With the described PCR condition of above-mentioned 7.1.10 part, by PCR the kpnI site is introduced among the streptomyces hygroscopicus DNA, its used primer to the right is 5 '-CTTCAGGTGTACGTGTTCG-3 ' (SEQ ID NO:23), and primer is 5 '-GAACTGGTACCAGTGCCC-3 ' (SEQ ID NO:24) (being provided by Genosys Biotechnologies company) left.The PCR product is digested with BsaAI and KpnI, separate these fragments, from gel, separate the BsaAI/KpnI fragment of 564bp by in 1% sepharose, carrying out electrophoresis.With KpnI and HindIII digestion, by carrying out electrophoresis separating these fragments in 1% sepharose, and fragment that will about 4.5Kb is separated from gel with pSE179 (as described in above-mentioned 7.1.10 part).With HindIII and BsaAl digestion pSE179, separate these fragments by in 1% sepharose, carrying out electrophoresis, and from gel, separate the BsaAI/HindIII fragment of an about 0.2Kb.The HindIII/kpnI fragment of this 4.5Kb, 0.2Kb BsaAl/HindIII fragment and the BsaAI/KpnI fragment that comes from 564 bp of streptomyces hygroscopicus link together in three kinds of modes, and this connection mixture transforms in the into competent E.coli DH5 α cell.From the amicillin resistance transformant, isolate plasmid DNA, confirm the segmental existence of this correction insertion by using KpnI and AvaI restricted enzyme cutting analysis.This plasmid digests to discharge 1.2Kb with HindIII and XbaI and inserts fragment, is connected with pWHM3 (it is with HindIII and XbaI digestion) then.Should connect mixture transforms in the into competent E.coli DH5 α cell.From the amicillin resistance transformant, isolate plasmid DNA, and confirm to proofread and correct the segmental existence of insertion with HindIII and AvaI restricted enzyme cutting analysis.This plasmid DNA transforms and enters among the E.coli DM1, isolates plasmid DNA from the amicillin resistance transformant, confirms to proofread and correct the segmental existence of insertion by restricted enzyme cutting analysis and dna sequence analysis.This plasmid is named as pSE350, and is used to transform the protoplastis of deinsectization streptomycete strain SE180-11.The thiostrepton resistance transformant of bacterial strain SE180-11 is separated, define the erythromycin resistance and exist, and analyze tunning by HPLC Thior Ermr transformant is analyzed.The result demonstrates the average B2 of the transformant that contains deinsectization streptomycete/streptomyces hygroscopicus heterozygosis plasmid: the B1 ratio is about 109: 1 (referring to table 6).
Table 6
Deinsectization streptomycete strain (conversion plasmid) The transformant Test No. The B2 relative concentration The B1 relative concentration Average B2: B1 ratio
SE180-11 (nothing) 8 ?0 ?0 ?0
SE180-11 (pWHM3) 8 ?0 ?0 ?0
SE180-11 (pSE350) 16 ?233 ?2 ?109
The preservation of biomaterial
On January 29th, 1998, following biomaterial is deposited in American type culture collection (ATCC), 12301 Parklawn Drive, and Rockville, MD, 20852, USA provides following preserving number:
The plasmid preserving number
Plasmid pSE180 209605
Plasmid pSE186 209604
Above whole patent of quoting, patent application, and open source literature is incorporated by reference comprehensively at this.
The present invention is not limited to the scope of specific embodiments described herein, and these are only given an example for the indivedual of all respects of the present invention, and method and composition suitable on the function all belongs to scope of the present invention.In fact, except above-mentioned explanation and example, from above-mentioned explanation and accompanying drawing, various variations of the present invention all are clearly to those skilled in the art.These variations all will fall within the protection domain of appended claims.
Sequence table
＜110〉Pfizer Products Inc's (non-U. S. application)
＜120〉mediation avermectin B2: the deinsectization streptomycete gene of B1 ratio
<130>PC9916A
<140>
<141>
<150>60/074,636
<151>1998-02-13
<160>24
<170>PatentIn?Ver.2.0-beta
<210>1
<211>1229
<212>DNA
＜213〉deinsectization streptomycete
<220>
<221>CDS
<222>(174)..(1085)
<400>1
tcacgaaacc?ggacacacca?cacacacgaa?ggtgagacag?cgtgaaccca?tccgagccgc??60
tcggcctgcc?caacgaacgt?gtagtagaca?cccgaccgtc?cgatgccacg?ctctcacccg??120
aggccggcct?gaacaggtca?ggagcgctgc?cccgtgaact?gctgtcgttg?ccg?gtg?????176
Val
1
gtg?gtg?tgg?gcc?ggg?gtc?ggc?ctg?ctg?ttt?ctg?gcc?ctg?cag?gcg?tac????224
Val?Val?Trp?Ala?Gly?Val?Gly?Leu?Leu?Phe?Leu?Ala?Leu?Gln?Ala?Tyr
5??????????????????10??????????????????15
gtg?ttc?agc?cgc?tgg?gcg?gcc?gac?ggt?ggc?tac?cgg?ctg?atc?gag?acg????272
Val?Phe?Ser?Arg?Trp?Ala?Ala?Asp?Gly?Gly?Tyr?Arg?Leu?Ile?Glu?Thr
20??????????????????25??????????????????30
gcg?ggc?cag?ggt?cag?ggc?ggc?agc?aag?gat?acg?ggg?act?acc?gat?gtg????320
Ala?Gly?Gln?Gly?Gln?Gly?Gly?Ser?Lys?Asp?Thr?Gly?Thr?Thr?Asp?Val
35??????????????????40??????????????????45
gtc?tat?ccc?gtg?att?tcc?gtc?gtc?tgc?atc?acc?gcc?gcg?gcg?gcg?tgg????368
Val?Tyr?Pro?Val?Ile?Ser?Val?Val?Cys?Ile?Thr?Ala?Ala?Ala?Ala?Trp
50??????????????????55??????????????????60??????????????????65
ctc?ttc?cgg?agg?tgc?cgt?gtc?gaa?cga?cgg?ctg?ctg?ttc?gac?gcc?ctt????416
Leu?Phe?Arg?Arg?Cys?Arg?Val?Glu?Arg?Arg?Leu?Leu?Phe?Asp?Ala?Leu
70??????????????????75??????????????????80
ctc?ttc?ctc?ggg?ctg?ctg?ttc?gcg?agc?tgg?cag?agc?ccg?ctc?atg?aac????464
Leu?Phe?Leu?Gly?Leu?Leu?Phe?Ala?Ser?Trp?Gln?Ser?Pro?Leu?Met?Asn
85??????????????????90??????????????????95
tgg?ttc?cat?tcc?gtt?ctc?gtc?tcc?aac?gcg?agt?gtg?tgg?ggc?gcg?gtg????512
Trp?Phe?His?Ser?Val?Leu?Val?Ser?Asn?Ala?Ser?Val?Trp?Gly?Ala?Val
100?????????????????105?????????????????110
ggt?tcc?tgg?ggt?ccg?tat?gtg?ccc?ggc?tgg?cag?ggg?gcg?ggc?ccg?ggt????560
Gly?Ser?Trp?Gly?Pro?Tyr?Val?Pro?Gly?Trp?Gln?Gly?Ala?Gly?Pro?Gly
115?????????????????120?????????????????125
gcg?gag?gcg?gaa?atg?ccg?ctg?gcg?tcg?gcc?tcc?gtc?tgc?atg?tcg?gct????608
Ala?Glu?Ala?Glu?Met?Pro?Leu?Ala?Ser?Ala?Ser?Val?Cys?Met?Ser?Ala
130?????????????????135?????????????????140?????????????????145
ctg?atc?gtc?acc?gtg?ctg?tgc?agc?aag?gca?ctg?ggg?tgg?atc?aag?gcc????656
Leu?Ile?Val?Thr?Val?Leu?Cys?Ser?Lys?Ala?Leu?Gly?Trp?Ile?Lys?Ala
150????????????????155??????????????????160
cgc?cgg?ccg?gca?tgg?cgg?acc?tgg?cgg?ctg?gtc?ctg?gcc?gtg?ttc?ttc????704
Arg?Arg?Pro?Ala?Trp?Arg?Thr?Trp?Arg?Leu?Val?Leu?Ala?Val?Phe?Phe
165?????????????????170?????????????????175
atc?ggc?atc?gtg?ctc?ggt?ctg?tcc?gag?ccg?ctg?ccg?tcc?gcc?tcc?ggg????752
Ile?Gly?Ile?Val?Leu?Gly?Leu?Ser?Glu?Pro?Leu?Pro?Ser?Ala?Ser?Gly
180?????????????????185?????????????????190
atc?agc?gta?tgg?gcc?aga?gcg?ctg?ccc?gag?gtg?acc?ttg?tgg?agt?ggc????800
Ile?Ser?Val?Trp?Ala?Arg?Ala?Leu?Pro?Glu?Val?Thr?Leu?Trp?Ser?Gly
195?????????????????200?????????????????205
gag?tgg?tac?cag?ttc?ccc?gtg?tat?cag?gcg?gtc?ggt?tcc?ggc?ctg?gtc????848
Glu?Trp?Tyr?Gln?Phe?Pro?Val?Tyr?Gln?Ala?Val?Gly?Ser?Gly?Leu?Val
210?????????????????215?????????????????220?????????????????225
tgc?tgc?atg?ctg?ggc?tcg?ctg?cgc?ttc?ttc?cgc?gac?gaa?cgc?gat?gag????896
Cys?Cys?Met?Leu?Gly?Ser?Leu?Arg?Phe?Phe?Arg?Asp?Glu?Arg?Asp?Glu
230?????????????????235?????????????????240
tcg?tgg?gtg?gaa?cgg?gga?gcc?tgg?cgg?ttg?ccg?caa?cgg?gca?gcg?aac????944
Ser?Trp?Val?Glu?Arg?Gly?Ala?Trp?Arg?Leu?Pro?Gln?Arg?Ala?Ala?Asn
245?????????????????250?????????????????255
tgg?gcg?cgt?ttc?ctc?gcc?gtg?gtc?ggt?ggg?gtg?aat?gcc?gtg?atg?ttc????992
Trp?Ala?Arg?Phe?Leu?Ala?Val?Val?Gly?Gly?Val?Asn?Ala?Val?Met?Phe
260?????????????????265?????????????????270
ctc?tac?acc?tgt?ttc?cat?atc?ctc?ctg?tcc?ctc?gtc?ggt?gga?cag?ccg????1040
Leu?Tyr?Thr?Cys?Phe?His?Ile?Leu?Leu?Ser?Leu?Val?Gly?Gly?Gln?Pro
275?????????????????280?????????????????285
ccc?gac?caa?ctg?ccg?gac?tcc?ttc?caa?gcg?ccg?gcc?gct?tac?tga????????1085
Pro?Asp?Gln?Leu?Pro?Asp?Ser?Phe?Gln?Ala?Pro?Ala?Ala?Tyr
290?????????????????295?????????????????300
gttcagggca?ggtcggagga?gacggagaag?gggaggcgac?cggagttccg?gtcacctccc??1145
ctttgtgcat?gggtggacgg?ggatcacgct?cccatggcgg?cgggctcctc?cagacgcacc??1205
acactcctcg?gttcagcgat?catg?????????????????????????????????????????1229
<210>2
<211>303
<212>PRT
＜213〉deinsectization streptomycete
<400>2
Val?Val?Val?Trp?Ala?Gly?Val?Gly?Leu?Leu?Phe?Leu?Ala?Leu?Gln?Ala
l???????????????5??????????????????10??????????????????15
Tyr?Val?Phe?Ser?Arg?Trp?Ala?Ala?Asp?Gly?Gly?Tyr?Arg?Leu?Ile?Glu
20??????????????????25??????????????????30
Thr?Ala?Gly?Gln?Gly?Gln?Gly?Gly?Ser?Lys?Asp?Thr?Gly?Thr?Thr?Asp
35??????????????????40??????????????????45
Val?Val?Tyr?Pro?Val?Ile?Ser?Val?Val?Cys?Ile?Thr?Ala?Ala?Ala?Ala
50??????????????????55??????????????????60
Trp?Leu?Phe?Arg?Arg?Cys?Arg?Val?Glu?Arg?Arg?Leu?Leu?Phe?Asp?Ala
65??????????????????70??????????????????75??????????????????80
Leu?Leu?Phe?Leu?Gly?Leu?Leu?Phe?Ala?Ser?Trp?Gln?Ser?Pro?Leu?Met
85??????????????????90??????????????????95
Asn?Trp?Phe?His?Ser?Val?Leu?Val?Ser?Asn?Ala?Ser?Val?Trp?Gly?Ala
100?????????????????105?????????????????110
Val?Gly?Ser?Trp?Gly?Pro?Tyr?Val?Pro?Gly?Trp?Gln?Gly?Ala?Gly?Pro
115?????????????????120?????????????????125
Gly?Ala?Glu?Ala?Glu?Met?Pro?Leu?Ala?Ser?Ala?Ser?Val?Cys?Met?Ser
130?????????????????135?????????????????140
Ala?Leu?Ile?Val?Thr?Val?Leu?Cys?Ser?Lys?Ala?Leu?Gly?Trp?Ile?Lys
145?????????????????150?????????????????155?????????????????160
Ala?Arg?Arg?Pro?Ala?Trp?Arg?Thr?Trp?Arg?Leu?Val?Leu?Ala?Val?Phe
165?????????????????170?????????????????175
Phe?Ile?Gly?Ile?Val?Leu?Gly?Leu?Ser?Glu?Pro?Leu?Pro?Ser?Ala?Ser
180?????????????????185?????????????????190
Gly?Ile?Ser?Val?Trp?Ala?Arg?Ala?Leu?Pro?Glu?Val?Thr?Leu?Trp?Ser
195?????????????????200?????????????????205
Gly?Glu?Trp?Tyr?Gln?Phe?Pro?Val?Tyr?Gln?Ala?Val?Gly?Ser?Gly?Leu
210?????????????????215?????????????????220
Val?Cys?Cys?Met?Leu?Gly?Ser?Leu?Arg?Phe?Phe?Arg?Asp?Glu?Arg?Asp
225?????????????????230?????????????????235?????????????????240
Glu?Ser?Trp?Val?Glu?Arg?Gly?Ala?Trp?Arg?Leu?Pro?Gln?Arg?Ala?Ala
245?????????????????250?????????????????255
Asn?Trp?Ala?Arg?Phe?Leu?Ala?Val?Val?Gly?Gly?Val?Asn?Ala?Val?Met
260?????????????????265?????????????????270
Phe?Leu?Tyr?Thr?Cys?Phe?His?Ile?Leu?Leu?Ser?Leu?Val?Gly?Gly?Gln
275?????????????????280?????????????????285
Pro?Pro?Asp?Gln?Leu?Pro?Asp?Ser?Phe?Gln?Ala?Pro?Ala?Ala?Tyr
290?????????????????295?????????????????300
<210>3
<211>1150
<212>DNA
＜213〉streptomyces hygroscopicus
<220>
<221>CDS
<222>(58)..(990)
<400>3
gtcgacgaag?accggccgga?ggccgtcggc?cgggccgata?ccgtacgcgg?cctgcgg?????57
gtg?ttc?acc?ctt?ccc?gta?aca?ctg?tgg?gcg?tgt?gtc?ggc?gcg?ctg?gtg????105
Val?Phe?Thr?Leu?Pro?Val?Thr?Leu?Trp?Ala?Cys?Val?Gly?Ala?Leu?Val
1???????????????5??????????????????10??????????????????15
ctg?gga?ctt?cag?gtg?tac?gtg?ttc?gcc?gcc?tgg?ctc?gcc?gac?agc?ggc????153
Leu?Gly?Leu?Gln?Val?Tyr?Val?Phe?Ala?Ala?Trp?Leu?Ala?Asp?Ser?Gly
20??????????????????25??????????????????30
tac?cgc?atc?gag?aag?gcg?tcc?ccg?gcc?agg?ggc?ggt?ggg?gac?tcg?gag????201
Tyr?Arg?Ile?Glu?Lys?Ala?Ser?Pro?Ala?Arg?Gly?Gly?Gly?Asp?Ser?Glu
35??????????????????40??????????????????45
cgg?atc?gcc?gat?gtg?ctg?atc?ccg?ctg?ctg?tcc?gtg?gtg?gga?gcg?gtg????249
Arg?Ile?Ala?Asp?Val?Leu?Ile?Pro?Leu?Leu?Ser?Val?Val?Gly?Ala?Val
50??????????????????55??????????????????60
gtc?ctc?gca?gtg?tgt?ctg?tac?cgg?agg?tgt?cgg?gcc?agg?agg?cgg?ctg????297
Va1?Leu?Ala?Val?Cys?Leu?Tyr?Arg?Arg?Cys?Arg?Ala?Arg?Arg?Arg?Leu
65??????????????????70??????????????????75??????????????????80
acg?ttc?gac?gcg?tcg?ctc?ttc?atc?ggg?ctg?ctg?tcg?gcc?agt?tgg?cag????345
Thr?Phe?Asp?Ala?Ser?Leu?Phe?Ile?Gly?Leu?Leu?Ser?Ala?Ser?Trp?Gln
85?????????????????90??????????????????95
agt?ccc?ttg?atg?aac?tgg?atc?aat?ccg?gtg?ctc?gcg?tca?aac?gtc?aat????393
Ser?Pro?Leu?Met?Asn?Trp?Ile?Asn?Pro?Val?Leu?Ala?Ser?Asn?Val?Asn
100?????????????????105?????????????????110
gtg?ttc?gga?gcg?gtg?gcc?tcg?tgg?ggg?ccg?tat?gtg?ccc?ggt?tgg?cag????441
Val?Phe?Gly?Ala?Val?Ala?Ser?Trp?Gly?Pro?Tyr?Val?Pro?Gly?Trp?Gln
115?????????????????120?????????????????125
ggg?gcg?ggg?gcg?cac?cag?gag?gcc?gag?ctg?ccg?ctg?gcg?acc?ctg?agc????489
Gly?Ala?Gly?Ala?His?Gln?Glu?Ala?Glu?Leu?Pro?Leu?Ala?Thr?Leu?Ser
130?????????????????135?????????????????140
atc?tgt?atg?acg?gcc?atg?atg?gcc?gcc?gtg?gcc?tgc?ggc?aag?ggc?atg????537
Ile?Cys?Met?Thr?Ala?Met?Met?Ala?Ala?Val?Ala?Cys?Gly?Lys?Gly?Met
145?????????????????150?????????????????155?????????????????160
ggt?ctt?gcc?gcc?gcc?cgg?tgg?ccg?cgg?ctg?ggg?ccg?ctc?cgg?ctg?atc????585
Gly?Leu?Ala?Ala?Ala?Arg?Trp?Pro?Arg?Leu?Gly?Pro?Leu?Arg?Leu?Ile
165?????????????????170?????????????????175
gcg?ctc?ggc?ttt?ctg?ctc?gtc?gtg?ctc?ctc?gac?atc?gcc?gag?ccg?ctg????633
Ala?Leu?Gly?Phe?Leu?Leu?Val?Val?Leu?Leu?Asp?Ile?Ala?Glu?Pro?Leu
180?????????????????185?????????????????190
gtg?tcc?ttc?gcg?ggc?gtc?tcc?gtg?tgg?acg?cgg?gca?gtg?ccc?gag?ctg????681
Val?Ser?Phe?Ala?Gly?Val?Ser?Val?Trp?Thr?Arg?Ala?Val?Pro?Glu?Leu
195?????????????????200?????????????????205
acc?atc?tgg?agt?ggg?cac?tgg?tat?cag?ttc?ccg?ctg?tat?cag?atg?gtg????729
Thr?Ile?Trp?Ser?Gly?His?Trp?Tyr?Gln?Phe?Pro?Leu?Tyr?Gln?Met?Val
210?????????????????215?????????????????220
gct?tcg?gcg?ctc?ttc?ggc?gcc?tct?ttg?ggg?gcc?gcg?cgc?cac?ttt?cgc????777
Ala?Ser?Ala?Leu?Phe?Gly?Ala?Ser?Leu?Gly?Ala?Ala?Arg?His?Phe?Arg
225?????????????????230?????????????????235?????????????????240
aac?cgg?cgc?ggc?gaa?acg?tgt?ctg?gag?tcc?ggg?gcg?gcc?ctc?cta?ccg????825
Asn?Arg?Arg?Gly?Glu?Thr?Cys?Leu?Glu?Ser?Gly?Ala?Ala?Leu?Leu?Pro
245?????????????????250?????????????????255
gag?ggc?ccg?agg?cca?tgg?gtc?cgg?ctg?ctg?gcg?gtg?gtg?ggc?ggg?gcc????873
Glu?Gly?Pro?Arg?Pro?Trp?Val?Arg?Leu?Leu?Ala?Val?Val?Gly?Gly?Ala
260?????????????????265?????????????????270
aac?atc?agc?atc?gcc?ctc?tac?acc?ggc?gca?cac?ggc?gca?cac?atc?ctg????921
Asn?Ile?Ser?Ile?Ala?Leu?Tyr?Thr?Gly?Ala?His?Gly?Ala?His?Ile?Leu
275?????????????????280?????????????????285
ttc?tcg?ctg?atg?gac?ggc?gct?ccc?ccg?gac?cgg?ctc?ccc?gaa?ttc?ttc????969
Phe?Ser?Leu?Met?Asp?Gly?Ala?Pro?Pro?Asp?Arg?Leu?Pro?Glu?Phe?Phe
290?????????????????295?????????????????300
cgt?ccg?gcg?gcc?ggc?tac?tga?gaccgccggc?accacccacg?tacccgatgt???????1020
Arg?Pro?Ala?Ala?Gly?Tyr
305?????????????????310
gcgcgatgtg?cctgatgcgc?ctgatgtacc?cggggtgtca?tcggctcacc?tgtggcgcct??1080
catgcggtga?gcgctccgcc?tcgtccttgt?tccggctcct?gggctccacg?accatacgga??1140
gcggccgggg?????????????????????????????????????????????????????????1150
<210>4
<211>310
<212>PRT
＜213〉streptomyces hygroscopicus
<400>4
Val?Phe?Thr?Leu?Pro?Val?Thr?Leu?Trp?Ala?Cys?Val?Gly?Ala?Leu?Val
1???????????????5??????????????????10??????????????????15
Leu?Gly?Leu?Gln?Val?Tyr?Val?Phe?Ala?Ala?Trp?Leu?Ala?Asp?Ser?Gly
20??????????????????25??????????????????30
Tyr?Arg?Ile?Glu?Lys?Ala?Ser?Pro?Ala?Arg?Gly?Gly?Gly?Asp?Ser?Glu
35??????????????????40??????????????????45
Arg?Ile?Ala?Asp?Val?Leu?Ile?Pro?Leu?Leu?Ser?Val?Val?Gly?Ala?Val
50??????????????????55??????????????????60
Val?Leu?Ala?Val?Cys?Leu?Tyr?Arg?Arg?Cys?Arg?Ala?Arg?Arg?Arg?Leu
65?????????????????70??????????????????75??????????????????80
Thr?Phe?Asp?Ala?Ser?Leu?Phe?Ile?Gly?Leu?Leu?Ser?Ala?Ser?Trp?GLn
85??????????????????90??????????????????95
Ser?Pro?Leu?Met?Asn?Trp?Ile?Asn?Pro?Val?Leu?Ala?Ser?Asn?Val?Asn
100?????????????????105?????????????????110
Val?Phe?Gly?Ala?Val?Ala?Ser?Trp?Gly?Pro?Tyr?Val?Pro?Gly?Trp?Gln
115?????????????????120?????????????????125
Gly?Ala?Gly?Ala?His?Gln?Glu?Ala?Glu?Leu?Pro?Leu?Ala?Thr?Leu?Ser
130?????????????????135?????????????????140
Ile?Cys?Met?Thr?Ala?Met?Met?Ala?Ala?Val?Ala?Cys?Gly?Lys?Gly?Met
145?????????????????150?????????????????155?????????????????160
Gly?Leu?Ala?Ala?Ala?Arg?Trp?Pro?Arg?Leu?Gly?Pro?Leu?Arg?Leu?Ile
165?????????????????170?????????????????175
Ala?Leu?Gly?Phe?Leu?Leu?Val?Val?Leu?Leu?Asp?Ile?Ala?Glu?Pro?Leu
180?????????????????185?????????????????190
Val?Ser?Phe?Ala?Gly?Val?Ser?Val?Trp?Thr?Arg?Ala?Val?Pro?Glu?Leu
195?????????????????200?????????????????205
Thr?Ile?Trp?Ser?Gly?His?Trp?Tyr?Gln?Phe?Pro?Leu?Tyr?Gln?Met?Val
210?????????????????215?????????????????220
Ala?Ser?Ala?Leu?Phe?Gly?Ala?Ser?Leu?Gly?Ala?Ala?Arg?His?Phe?Arg
225?????????????????230?????????????????235?????????????????240
Asn?Arg?Arg?Gly?Glu?Thr?Cys?Leu?Glu?Ser?Gly?Ala?Ala?Leu?Leu?Pro
245?????????????????250?????????????????255
Glu?Gly?Pro?Arg?Pro?Trp?Val?Arg?Leu?Leu?Ala?Val?Val?Gly?Gly?Ala
260?????????????????265?????????????????270
Asn?Ile?Ser?Ile?Ala?Leu?Tyr?Thr?Gly?Ala?His?Gly?Ala?His?Ile?Leu
275?????????????????280?????????????????285
Phe?Ser?Leu?Met?Asp?Gly?Ala?Pro?Pro?Asp?Arg?Leu?Pro?Glu?Phe?Phe
290?????????????????295?????????????????300
Arg?Pro?Ala?Ala?Gly?Tyr
305?????????????????310
<210>5
<211>215
<212>PRT
＜213〉ash produces the look streptomycete
<400>5
Val?Ile?Gly?Trp?Ala?Ala?Leu?Gly?Ala?Val?Phe?Leu?Val?Leu?Gln?Val
1???????????????5??????????????????10??????????????????15
Tyr?Val?Phe?Ala?Arg?Trp?Thr?Ala?Asp?Gly?Gly?Tyr?His?Leu?Ala?Asp
20??????????????????25??????????????????30
Val?Ser?Gly?Pro?Asp?Gly?Arg?Glu?Pro?Gly?His?Arg?Arg?Ile?Ile?Asp
35??????????????????40??????????????????45
Val?Leu?Leu?Pro?Ala?Leu?Ser?Met?Ala?Gly?Val?Val?Gly?Leu?Ala?Phe
50??????????????????55??????????????????60
Trp?Leu?Val?Arg?Arg?Trp?Arg?Ala?Glu?Arg?Arg?Leu?Ser?Phe?Asp?Ala
65??????????????????70??????????????????75??????????????????80
Leu?Leu?Phe?Thr?Gly?Val?Leu?Phe?Ala?Gly?Trp?Leu?Ser?Pro?Leu?Met
85??????????????????90??????????????????95
Asn?Trp?Phe?His?Pro?Val?Leu?Met?Ala?Asn?Thr?His?Val?Trp?Gly?Ala
100?????????????????105?????????????????110
Val?Gly?Ser?Trp?Gly?Pro?Tyr?Val?Pro?Gly?Trp?Arg?Gly?Leu?Pro?Pro
115?????????????????120?????????????????125
Gly?Lys?Glu?Ala?Glu?Leu?Pro?Leu?Val?Thr?Phe?Ser?Leu?Gly?Ser?Thr
130?????????????????135?????????????????140
Val?Leu?Leu?Gly?Val?Leu?Gly?Cys?Cys?Gln?Val?Met?Ser?Arg?Val?Arg
145?????????????????150?????????????????155?????????????????160
Glu?Arg?Trp?Pro?Gly?Val?Arg?Pro?Trp?Gln?Leu?Val?Gly?Leu?Ala?Phe
165?????????????????170?????????????????175
Leu?Thr?Ala?Val?Ala?Phe?Asp?Leu?Ser?Glu?Pro?Phe?Ile?Ser?Phe?Ala
180?????????????????185?????????????????190
Gly?Val?Ser?Val?Trp?Ala?Arg?Ala?Leu?Pro?Thr?Val?Thr?Leu?Trp?Arg
195?????????????????200?????????????????205
Gly?Ala?Trp?Tyr?Arg?Ala?Arg
210?????????????????215
<210>6
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>6
tcacgaaacc?ggacacac??????????????????????????????????????????????18
<210>7
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>7
catgatcgct?gaaccgag??????????????????????????????????????????????18
<210>8
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>8
ggttccggat?gccgttctcg????????????????????????????????????????????20
<210>9
<211>21
<212>DNA
＜213〉deinsectization streptomycete
<400>9
aactccggtc?gactcccctt?c??????????????????????????????????????????21
<210>10
<211>19
<212>DNA
＜213〉deinsectization streptomycete
<400>10
gcaaggatac?ggggactac?????????????????????????????????????????????19
<210>11
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>11
gaaccgaccg?cctgatac??????????????????????????????????????????????18
<210>12
<211>43
<212>DNA
＜213〉deinsectization streptomycete
<400>12
gggggcgggc?ccgggtgcgg?aggcggaaat?gcccctggcg?acg??????????????????43
<210>13
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>13
ggaaccgacc?gcctgataca????????????????????????????????????????????20
<210>14
<211>46
<212>DNA
＜213〉deinsectization streptomycete
<400>14
gggggcgggc?ccgggtgcgg?aggcggaaat?gccgctggcg?acgacc??????????????46
<210>15
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>15
ggaacatcac?ggcattcacc???????????????????????????????????????????20
<210>16
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>16
aacccatccg?agccgctc?????????????????????????????????????????????18
<210>17
<211>17
<212>DNA
＜213〉deinsectization streptomycete
<400>17
tcggcctgcc?aacgaac??????????????????????????????????????????????17
<210>18
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>18
ccaacgaacg?tgtagtag?????????????????????????????????????????????18
<210>19
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>19
tgcaggcgta?cgtgttcagc???????????????????????????????????????????20
<210>20
<211>17
<212>DNA
＜213〉deinsectization streptomycete
<400>20
catgatcgct?gaaccga??????????????????????????????????????????????17
<210>21
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>21
catgatcgct?gaaccgagga???????????????????????????????????????????20
<210>22
<211>20
<212>DNA
＜213〉deinsectization streptomycete
<400>22
aggagtgtgg?tgcgtctgga???????????????????????????????????????????20
<210>23
<211>19
<212>DNA
＜213〉deinsectization streptomycete
<400>23
cttcaggtgt?acgtgttcg???????????????????????????????????????????????19
<210>24
<211>18
<212>DNA
＜213〉deinsectization streptomycete
<400>24
gaactggtac?cagtgccc????????????????????????????????????????????????18

Claims (71)

1. isolating polynucleotide molecule, it comprises the complete aveC ORF of deinsectization streptomycete or its substantive part, and this isolating polynucleotide molecule lacks the next complete ORF that is arranged in deinsectization streptomycete karyomit(e) aveC ORF original position downstream.
2. isolating polynucleotide molecule as claimed in claim 1, it comprises the identical nucleotide sequence of deinsectization streptomycete AveC gene product encoding sequence with plasmid pSE186 (ATCC 209604), perhaps with aveC ORF nucleotide sequence or the identical nucleotide sequence of its substantive part shown in Fig. 1 (SEQ ID NO:1).
3. isolating polynucleotide molecule as claimed in claim 2, it further comprises the nucleotide sequence of the original position AveC gene of natural flank in deinsectization streptomycete.
4. isolating polynucleotide molecule, it comprises the deinsectization streptomycete AveC gene product encoding sequence with plasmid pSE186 (ATCC 209604), perhaps the nucleotide sequence that has homology with the nucleotide sequence of the aveCORF shown in Fig. 1 (SEQ ID NO:1) or its substantive part.
5. isolating polynucleotide molecule, it comprises the nucleotide sequence of coded polypeptide, the coded aminoacid sequence of AveC gene product encoding sequence of aminoacid sequence that described polypeptide has and plasmid pSE186 (ATCC 209604) perhaps has homology with the aminoacid sequence shown in Fig. 1 (SEQ ID NO:2) or its substantive part.
6. isolating polynucleotide molecule, it comprises nucleotide sequence or its substantive part of SEQ ID NO:3, perhaps the nucleotide sequence that has homology with the nucleotide sequence of SEQ ID NO:3.
7. isolating polynucleotide molecule, it comprises the nucleotide sequence of coded polypeptide, the aminoacid sequence of this polypeptide and SEQ ID NO:4 has homology.
8. oligonucleotide molecules, it is under highly tight condition, hybridize with the polynucleotide molecule of nucleotide sequence, perhaps hybridize with the polynucleotide molecule that has with the nucleotide sequence complementary nucleotide sequence of Fig. 1 (SEQ ID NO:1) or SEQ ID NO:3 with Fig. 1 (SEQ ID NO:1) or SEQ ID NO:3.
9. oligonucleotide molecules as claimed in claim 8, the nucleotide sequence complementation of itself and Fig. 1 (SEQ ID NO:1) or SEQ ID NO:3 is perhaps with the polynucleotide molecule complementation that has with the nucleotide sequence complementary nucleotide sequence of table 1 (SEQ ID NO:1) or SEQ ID NO:3.
10. recombinant vectors, it comprises polynucleotide molecule, this polynucleotide molecule contains and is selected from following nucleotide sequence:
(a) nucleotide sequence, its deinsectization streptomycete AveC gene product encoding sequence with plasmid pSE186 (ATCC 209604) is identical, and perhaps nucleotide sequence or its substantive part with the aveC ORF of Fig. 1 (SEQ ID NO:1) is identical;
(b) nucleotide sequence, the deinsectization streptomycete AveC gene product encoding sequence of itself and plasmid pSE186 (ATCC 209604), perhaps nucleotide sequence or its substantive part with the aveC ORF of Fig. 1 (SEQ ID NO:1) has homology;
(c) nucleotide sequence, the coded aminoacid sequence of AveC gene product encoding sequence of aminoacid sequence that its encoded polypeptides has and plasmid pSE186 (ATCC 209604), perhaps aminoacid sequence or its substantive part with Fig. 1 (SEQ ID NO:2) has homology.
11. recombinant vectors as claimed in claim 10, it further comprises the nucleotide sequence of one or more controlling elements of encoding, and the nucleotide sequence of this coding controlling element can be operated with nucleotide sequence as claimed in claim 10 and link to each other.
12. recombinant vectors as claimed in claim 11, it further comprises the nucleotide sequence of the selected marker of encoding.
13. recombinant vectors as claimed in claim 12, it is plasmid PSE186 (ATCC209604).
14. comprise the host cell of recombinant vectors as claimed in claim 12.
15. host cell as claimed in claim 14, it is a deinsectization streptomycete.
16. recombinant vectors, it comprises the polynucleotide molecule that contains nucleotide sequence, described nucleotide sequence is selected from following sequence: the nucleotide sequence of SEQ ID NO:3 or its substantive part, with the nucleotide sequence that the nucleotide sequence shown in the SEQID NO:3 has homology, the aminoacid sequence shown in coding and the SEQ IDNO:4 has the nucleotide sequence of the polypeptide of homology.
17. recombinant vectors as claimed in claim 16 further comprises the nucleotide sequence of one or more controlling elements of encoding, the nucleotide sequence of this coding controlling element can be operated with nucleotide sequence as claimed in claim 16 and link to each other.
18. recombinant vectors as claimed in claim 17 further comprises the nucleotide sequence of the selected marker of encoding.
19. comprise the host cell of recombinant vectors as claimed in claim 18.
20. host cell as claimed in claim 19, it is a streptomyces hygroscopicus.
21. recombinant expressed deinsectization streptomycete AveC gene product or its homology polypeptide, described gene product has the coded aminoacid sequence of deinsectization streptomycete AveC gene product coding nucleotide sequence by plasmid pSE186 (ATCC 209604), perhaps has the aminoacid sequence shown in the SEQ ID NO:2 or its substantive part.
22. recombinant expressed streptomyces hygroscopicus AveC homologue gene product or its homeopeptide, described gene product has aminoacid sequence shown in the SEQ ID NO:4.
23. be used to produce the method for reorganization AveC gene product, it is included in to cultivate under the condition that is beneficial to the production of reorganization AveC gene product uses the recombinant expression vector transformed host cells, and from cell culture, reclaim the AveC gene product, described recombinant expression vector comprises the polynucleotide molecule with nucleotide sequence, the described nucleotide sequence coded coded aminoacid sequence of deinsectization streptomycete AveC gene product coding nucleotide sequence by plasmid pSE186 (ATCC 209604), perhaps the encode aminoacid sequence shown in the SEQ IDNO:2 or its substantive part, described polynucleotide molecule can be operated with the controlling element of the expression of one or more control polynucleotide molecules in host cell and link to each other.
24. be used to produce the method for reorganization AveC homologue gene product, it is included in to cultivate under the condition that is beneficial to the production of reorganization AveC homologue gene product uses the recombinant expression vector transformed host cells, and from culture, reclaim AveC homologue gene product, described recombinant expression vector comprises the polynucleotide molecule with nucleotide sequence, aminoacid sequence or its substantive part shown in the described nucleotide sequence coded SEQ ID NO:4, described polynucleotide molecule can be operated with the controlling element of the expression of one or more control polynucleotide molecules in host cell and be linked to each other.
25. polynucleotide molecule, the nucleotide sequence that it has or identical with the deinsectization streptomycete AveC gene product encoding sequence of plasmid pSE186 (ATCC209604), perhaps the nucleotide sequence with the aveC ORF of deinsectization streptomycete shown in Fig. 1 (SEQ IDNO:1) is identical; But this nucleotide sequence further comprises one or more sudden changes, thereby makes that the ratio and the amount of the avermectin that the cell of deinsectization streptomycete bacterial strain ATCC53692 of polynucleotide molecule that inactivation and the expression of wild-type aveC allelotrope wherein contains the nucleotide sequence of sudden change produces are different with the ratio that cell produced of only expressing the allelic deinsectization streptomycete bacterial strain ATCC 53692 of wild-type aveC and amount.
26. polynucleotide molecule as claimed in claim 25, the production of its modulation avermectin, make wild-type aveC allelotrope wherein inactivation and the cell of expressing the deinsectization streptomycete bacterial strain ATCC 53692 of the described polynucleotide molecule of claim 25 compare 2 classes to reduce with the cell of only expressing the allelic deinsectization streptomycete bacterial strain ATCC 53692 of wild-type aveC: 1 class ratio produces avermectin.
27. polynucleotide molecule as claimed in claim 26, wherein 2 classes: 1 class avermectin is cyclohexyl B2: cyclohexyl B1 avermectin.
28. polynucleotide molecule as claimed in claim 27, wherein the cyclohexyl B2 of Jiang Diing: cyclohexyl B1 ratio was less than 1.6: 1.
29. polynucleotide molecule as claimed in claim 28, wherein the cyclohexyl B2 of Jiang Diing: cyclohexyl B1 ratio is about 0.94: 1.
30. polynucleotide molecule as claimed in claim 29, wherein residue 139 places of the sudden change of the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) coding AveC gene product are become the aminoacid replacement of Threonine by L-Ala.
31. polynucleotide molecule as claimed in claim 28, wherein the cyclohexyl B2 of Jiang Diing: the ratio of cyclohexyl B1 is approximately 0.88: 1.
32. polynucleotide molecule as claimed in claim 31, wherein residue 138 places of the sudden change of the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) coding AveC gene product are become the aminoacid replacement of Threonine by Serine.
33. polynucleotide molecule as claimed in claim 28, wherein the cyclohexyl B2 of Jiang Diing: cyclohexyl B1 ratio is approximately 0.84: 1.
34. polynucleotide molecule as claimed in claim 33, wherein residue 139 places that become first aminoacid replacement of Threonine and AveC gene product by Serine, residue 138 places of the sudden change of the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) coding AveC gene product are become second aminoacid replacement of Threonine by L-Ala.
35. polynucleotide molecule as claimed in claim 25, wherein residue 55 places of the sudden change of the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) coding AveC gene product are become the aminoacid replacement of phenylalanine by Serine.
36. polynucleotide molecule as claimed in claim 25, wherein residue 230 places of the sudden change of the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) coding AveC gene product are become the aminoacid replacement of aspartic acid by glycine.
37. contain the polynucleotide molecule of strong promoter, described promotor can be operated with the deinsectization streptomycete AveC ORF shown in Fig. 1 (SEQ ID NO:1) and be linked to each other.
38. polynucleotide molecule as claimed in claim 37, wherein said strong promoter are the ermE promotors from many spores of redness bacterium.
39. polynucleotide molecule, the nucleotide sequence of the coding AveC gene product that it comprises are by heterologous nucleotide sequence being inserted in the described nucleotide sequence and inactivation.
40. polynucleotide molecule as claimed in claim 25, it comprises aveC allelotrope, and this equipotential gene is inactivation by the PstI/SphI segment of disappearance 640bp from the AveC ORF of Fig. 1 (SEQ ID NO:1).
41. polynucleotide molecule as claimed in claim 25, it comprises aveC allelotrope, and this equipotential gene is by importing phase shift mutation and inactivation in this equipotential gene.
42. polynucleotide molecule as claimed in claim 41 wherein imports phase shift mutation by increase by two G Nucleotide behind the C Nucleotide at 471 Nucleotide places of the aveC ORF of Fig. 1 (SEQ IDNO:1).
43. polynucleotide molecule as claimed in claim 25, it comprises aveC allelotrope, wherein imports terminator codon by the nucleotide position place in the 116th amino acids of coding deinsectization streptomycete AveC gene product and makes this equipotential gene inactivation.
44. polynucleotide molecule as claimed in claim 25, it comprises aveC allelotrope, wherein import first sudden change that becomes aspartic acid by glycine, and become second sudden change of Histidine and make the aveC allelic inactivation by tyrosine in the importing of amino acid position 275 places of AveC gene product by amino acid position 258 places in the AveC gene product.
45. the gene replacement vector, it polynucleotide molecule that comprises contains the nucleotide sequence of the original position AveC gene of natural flank in deinsectization streptomycete karyomit(e).
46. recombinant vectors, it comprises arbitrary described polynucleotide molecule as claim 25-44.
47. recombinant vectors, it contains polynucleotide molecule as claimed in claim 39, and described carrier is pSE180 (ATCC 209605).
48. contain the streptomycete host cell of recombinant vectors as claimed in claim 46.
49. differentiate among the aveC ORF and can change avermectin 2 classes that produced: the method for the sudden change of 1 class ratio, described method comprises: (a) measure avermectin 2 classes that the deinsectization streptomycete bacterial strain cell produces: the ratio of 1 class, wild-type aveC allelotrope in the described cell is inactivation, and the polynucleotide molecule of nucleotide sequence that includes the AveC gene product of encoding mutant is imported into this cell and is expressed therein; (b) measure avermectin 2 classes of the strain cell generation identical with the deinsectization streptomycete of step (a): the ratio of 1 class, but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ 1D NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; Reach (c) avermectin 2 classes of the deinsectization streptomycete bacterial strain cell generation of comparison step (a): avermectin 2 classes that the deinsectization streptomycete bacterial strain cell of the ratio of 1 class and step (b) produces: the ratio of 1 class, if the two difference then can confirm to change avermectin 2 classes: the aveC ORF sudden change of 1 class ratio exists.
50. method as claimed in claim 49, wherein avermectin 2 classes: the ratio of 1 class reduces because of sudden change.
51. identify aveC ORF or include the method that can change the sudden change of the avermectin amount that is produced in the construct of aveC ORF, described method comprises: the amount of (a) measuring the avermectin of deinsectization streptomycete bacterial strain cell generation, wild-type aveC allelotrope in the described cell is inactivation, includes the nucleotide sequence of AveC gene product of encoding mutant or the polynucleotide molecule of gene construct that contains the nucleotide sequence of coding AveC gene product and is imported into this cell and expresses therein; (b) measure the amount of the avermectin of the strain cell generation identical with the deinsectization streptomycete of step (a), but different be this cell only express nucleotide sequence with the ORF shown in Fig. 1 (SEQ ID NO:1) or with the aveC allelotrope of its homologous nucleotide sequence; And (c) amount of amount and the avermectin of the deinsectization streptomycete bacterial strain cell generation of step (b) of the avermectin that produces of the deinsectization streptomycete bacterial strain cell of comparison step (a), if the two difference can confirm that then the aveC ORF or the gene construct sudden change that can change the avermectin amount exist.
52. method as claimed in claim 51, wherein the amount of the avermectin that is produced increases because of sudden change.
53. prepare the method for the new bacterial strain of deinsectization streptomycete, described bacterial strain includes the allelic cell of the aveC that can express sudden change, its with only express the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC and compare, 2 classes of the avermectin that is produced: 1 class ratio changes, described method comprises with the allelic carrier conversion of the aveC that carries sudden change deinsectization streptomycete bacterial strain cell, the gene product of the aveC allelotrope of this sudden change coding make the allelic deinsectization streptomycete bacterial strain cell of aveC of expressing sudden change with only express the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC and compare, 2 classes of the avermectin that is produced: 1 class ratio changes, select transformant, avermectin 2 classes that this transformant produces: 1 class ratio is more only expressed the allelic strain cell of wild-type aveC and is changed to some extent.
54. method as claimed in claim 53, wherein avermectin 2 classes: the ratio of 1 class reduces because of sudden change.
55. prepare the method for the new bacterial strain of deinsectization streptomycete, described bacterial strain comprises the cell of the avermectin that can produce the change amount, described method comprises with containing sudden change aveC allelotrope or containing the allelic gene construct conversion of this aveC deinsectization streptomycete bacterial strain cell, the amount that the avermectin amount that the result of its expression is produced the deinsectization streptomycete bacterial strain cell of expression sudden change aveC allelotrope or gene construct is more only expressed the avermectin of the identical strain cell generation of the allelic deinsectization streptomycete of wild-type aveC changes to some extent, select cell transformed, the avermectin amount that the avermectin amount that this transformant produces is more only expressed the allelic strain cell generation of wild-type aveC changes to some extent.
56. method as claimed in claim 55, wherein the amount of the avermectin that is produced increases because of sudden change.
57. prepare the method for the new bacterial strain of deinsectization streptomycete, described strain cell contains the aveC allelotrope of inactivation, described method comprises with the carrier that makes this aveC allelic inactivation and transforms the deinsectization streptomycete bacterial strain cell, and selects wherein the aveC allelotrope transformant of inactivation.
58. method as claimed in claim 57, wherein carrier is pSE 180 (ATCC209605).
59. deinsectization streptomycete bacterial strain, it contains the allelic cell of aveC of expressing sudden change, the result produces 2 classes: the deinsectization streptomycete cell that 1 class ratio changes to some extent, described 2 classes: the change of 1 class ratio is for only expressing the allelic identical strain cell of wild-type aveC.
60. bacterial strain as claimed in claim 59, wherein avermectin 2 classes: the ratio of 1 class reduces because of sudden change.
61. cell strain as claimed in claim 60, the cyclohexyl B2 that wherein cell produced: the ratio of cyclohexyl B1 avermectin was less than 1.6: 1.
62. bacterial strain as claimed in claim 61, the cyclohexyl B2 that wherein cell produced: the ratio of cyclohexyl B1 avermectin approximately is 0.94: 1.
63. bacterial strain as claimed in claim 61, the cyclohexyl B2 that wherein cell produced: the ratio of cyclohexyl B1 avermectin approximately is 0.88: 1.
64. bacterial strain as claimed in claim 61, the cyclohexyl B2 that wherein cell produced: the ratio of cyclohexyl B1 avermectin approximately is 0.84: 1.
65. deinsectization streptomycete bacterial strain, it contains the aveC allelotrope of expression sudden change or the cell of gene construct, the avermectin that increases to some extent of generation as a result, described amount increase is for only expressing the allelic identical strain cell of wild-type aveC.
66. deinsectization streptomycete bacterial strain, it contains the aveC gene cell of inactivation.
67. produce the method for avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim avermectin, the aveC allelotrope of described cell expressing sudden change, only express the allelic identical strain cell of aveC of wild-type with not expressing sudden change aveC allelotrope and compare, the gene product of this mutation allele coding has changed 2 classes of the avermectin of the allelic deinsectization streptomycete bacterial strain cell generation of the aveC that expresses sudden change: the ratio of 1 class.
68. as the described method of claim 67,2 classes of avermectin wherein: the ratio of 1 class reduces because of sudden change.
69. produce the method for avermectin, described method comprises: under the condition that allows or induce avermectin to produce, in substratum, cultivate the deinsectization streptomycete bacterial strain cell, and from culture, reclaim avermectin, the aveC allelotrope of described cell expressing sudden change or contain the allelic gene construct of aveC, only express the allelic identical strain cell of aveC of wild-type with not expressing sudden change aveC allelotrope or gene construct and compare, the amount of the avermectin that the aveC allelotrope of expression sudden change or the deinsectization streptomycete bacterial strain cell of gene construct produce changes to some extent.
70. as the described method of claim 69, wherein the amount of the avermectin that is produced increases by the aveC allelotrope or the gene construct of expressing sudden change.
71. the avermectin composition that is produced by the deinsectization streptomycete bacterial strain cell, the aveC allelotrope of the sudden change of described cell expressing encoding gene product, described gene product has reduced avermectin 2 classes that the aveC allelic deinsectization streptomycete bacterial strain cell of expressing sudden change is produced: 1 class ratio, described avermectin 2 classes: the minimizing of 1 class ratio is only to express with respect to the aveC allelotrope of not expressing sudden change for the allelic identical strain cell of wild-type aveC, wherein only express avermectin 2 classes that the identical strain cell of the allelic deinsectization streptomycete of wild-type aveC is produced with respect to the aveC allelotrope of not expressing sudden change: for the 1 class ratio, with 2 classes that reduce: 1 class ratio is produced avermectin.
CNA2004100054154A 1998-02-13 1999-01-25 Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins Pending CN1521180A (en)

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