CN102241750A - Genetic engineering method for producing avermectin and special bacterial strain for the method - Google Patents

Abstract

The invention discloses a genetic engineering method for producing avermectin and a special bacterial strain for the method. A protein provided by the invention is a mutant HrdB protein, which is specifically a protein 1) or 2) as follows: 1) a protein consisting of an amino acid sequence shown as sequence 4 in the sequence table; 2) a protein related to production of avermectin from streptomyces avermitilis and derived from 1) by substituting and / or deleting and / or adding one or a plurality of amino acid residues. The mutant HrdB gene provided by the invention is introduced into a ZLX6003 bacterial strains to obtain recombinant strains; after 240h of shaking culture of the recombinant strains, avermectin output can reach 5732.05+/-91.26 mug/ml. A growth curve of the engineering bacteria ZLX6056 in a 180t fermenter shows that the genetic engineering bacteria maintains excellent properties of a former high-yield bacterial strain to reach an output of 6382 mug/ml.

Description

A kind of gene engineering method and special strain therefore thereof of producing Avrmectin

Technical field

The present invention relates to a kind of gene engineering method and special strain therefore thereof of producing Avrmectin.

Background technology

(avermectin is a kind of macrolide antibiotics that is produced by Avid kyowamycin (Streptomyces avermitilis) fermentation AVM) to Avrmectin, and it has eight component (A1a, A1b, A2a, A2b, B1a, B1b, B2a, B2b), wherein the insecticidal activity of B1a component is the strongest, nematode, acarid and arthropods are had good killing action, and insecticidal activity exceeds tens times than general chemical pesticide.The mechanism of action of Avrmectin is different with the conventional chemical sterilant, and its action target spot is the parasitic nerve conduction medium γ-An Jidingsuan of nematode and arthropods class (GABA), and very little to mammiferous toxicity, selectivity is high.This microbiotic residence time in plant is very short, and very fast in soil be non-toxic substance by microbiological degradation.Because these outstanding advantages of Avrmectin, it is acknowledged as a kind of the most microbe-derived sterilant, thus it agricultural, forestry, medicine and for animals on have boundless market outlook and using value.Be parent with the Avrmectin simultaneously, can also develop the new variety of deriving that a series of activity are higher, selectivity is stronger, use is safer, commercial kind comprises ivermectin, emaricin, doractin, Ai Polinuo rhzomorph and Sai La rhzomorph etc.In addition, there is the investigator to find that avermitilis strain have antineoplastic effect in recent years again, and the resistance of tumour cell is also had certain restraining effect.

At present, Avrmectin has been realized industrialization at home, has obtained huge economic and social benefit.But it is low that the production bacterial strain of China's Avrmectin also exists fermentation unit, and how problems such as production cost height improve the output of Avrmectin, reduces production costs, and will play promoter action for the agriculture production development of China.In addition, be not only Avrmectin, how screening and optimizing bacterial strain, make productive rate and raw material availability realize maximizing is the common problem that China's microbial fermentation engineering faces.The method that is used to improve the microbiotic output of streptomycete at present mainly comprises two big classes, and a class is traditional method for mutation breeding, by physico-chemical process bacterial strain is carried out mutagenesis, screens superior strain then in mutagenic progeny; Another kind of is by genetic method, and bacterial strain is carried out direct hereditary change, improves antibiotic output.Obtained certain achievement though bacterial strain is carried out mutagenesis by physico-chemical process, obtained a large amount of superior strains, but their weak point also clearly, mainly comprise and expend a large amount of human and material resources and time, the screening operation more complicated, have certain blindness, when introducing favorable variation, may also can produce a lot of deleterious variations, and the variation of deleterious bacterial classification tends to influence the optimization and the amplification of fermenting process; These traditional strain improvement modes are not owing to understand the Basic of Biology that causes changes of function, and therefore difficulty is applied to other bacterial classifications.Along with deepening continuously of bacterial genomes research, constantly deep to the understanding of bacterial gene function, people more and more tend to the genetics means bacterial strain be transformed.The known gene of function can be transformed with genetic method, strengthen the specific aim of operation, and workload is reduced greatly, screening time also obtains shortening.Therefore, transform Avid kyowamycin to improve the output of Avrmectin, have great importance by genetic engineering means.

Calendar year 2001 Kitasato institute has finished the gene order-checking of Avid kyowamycin, carries out sequence and functional analysis to wherein being responsible for the Avrmectin biological synthesis gene cluster, and this gene cluster total length 82kb has 18 open reading frames.There are 4 big reading frames (AveA1-AveA2 and AveA3-AveA4), multifunctional coded polyketide synthetic enzyme in gene cluster inside; AveC and aveE gene are between aveA1-aveA2 and aveA3-aveA4 gene, and be relevant with the modification of polyketide; In the upstream of the contiguous aveA4 in the right side of gene cluster, be 8 genes (aveB I-aveBV III) that a cover relates to the synthetic of olea disaccharides and shifts; The upstream (left) of next-door neighbour aveA1 is the aveD of coding C5-O-methyltransgerase, and the responsible OH that methyl is given to the C5 of Avrmectin B goes up and formation Avrmectin A.The downstream of aveF next-door neighbour aveD, the two transcriptional orientation unanimity may belong to same transcription unit.AveF coding C5 keto reductase, the generation of catalysis Avrmectin B.AveR is positioned at the downstream (but transcriptional orientation opposite) of aveF, belongs to approach specificity regulatory gene, is the positive regulating gene of the full gene cluster of Avrmectin biosynthesizing.

The molecular regulation of gene level plays crucial effects in the microbiotic production process, by genetic method streptomycete is transformed, and improves microbiotic output.Mainly be to be undertaken, not only but also regulatory gene that with form differentiation be correlated with relevant with the microbiotic biosynthesizing arranged by transforming the regulatory gene that microbiotic produces, as bldA, relC, relA etc.; The biosynthetic regulatory gene of overall importance of the microbiotic of participation is also arranged, as absA, absB, afsR etc.; Participate in the biosynthetic specificity regulatory gene of microbiotic in addition, as ActII-ORF4, aveR, dnrI, redD, sanG, ccaR etc.Biosynthesizing plays the gene of positive control to microbiotic, can by improve it in bacterial strain copy number or change promotor and improve its expression amount and improve antibiotic output, to playing the gene of down regulation, then can improve antibiotic output by knocking out this gene.But, the bacterial strain that gets by the method that improves copy number is less stable often, because the Genetic carrier of high copy is often unstable, lose easily, if consider the industrial production in later stage, may more trials be incorporated on the karyomit(e) the more stable stable existence of high yield genes involved of keeping at engineering strain by homologous recombination.The regulatory gene that can regulate and control multiple microbiotic output is simultaneously transformed more effective than transforming the regulatory gene of only regulating and control single microbiotic output.

In Avid kyowamycin, to accessing the mutant strain that doractin " 1 " component concentration improves behind the aveC random mutation; To approach specificity regulatory gene aveR, increase copy number and do not improve Avrmectin output, make host strain no longer produce Avrmectin on the contrary.Have negative regulator gene aveR1 and aveR2 in the upstream of aveR gene, any one gene in these two genes or two gene inactivations simultaneously all can make Avrmectin output be greatly improved.(U.S. Pat 6197591, Stutzman-Engwall).Be arranged in outside the regulatory gene of Avrmectin biological synthesis gene cluster, the researchist also is devoted to seek other regulatory gene, regulatory gene particularly of overall importance (global regulatorygene) as afsR2 and orfX, thereby more effectively improves the output of Avrmectin.

Summary of the invention

The object of the present invention is to provide a kind of and Avid kyowamycin to produce the relevant albumen of Avrmectin.

Albumen provided by the invention, be the sudden change after HrdB albumen, specifically be following 1) or 2) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

2) with the amino acid residue sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with Avid kyowamycin produce Avrmectin relevant by 1) deutero-protein.

In order to make 1) in albumen be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 4 is formed in by sequence table or C-terminal connect.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) but in the albumen synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) the proteic encoding gene in can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 3, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

Above-mentioned proteic encoding gene (the hrdB gene after the sudden change) also belongs within protection scope of the present invention.

Said gene can be following 1) or 2) or 3):

1) encoding sequence is shown in sequence in the sequence table 3;

2) under stringent condition with 1) gene recombination and the gene of encoding said proteins;

3) with 1) gene have the homology more than 90% and the gene of encoding said proteins.

Sequence 3 is sequences (1539 Nucleotide) of the hrdB gene after the sudden change, the hrdB albumen in the codified sequence table after the sudden change shown in the sequence 4.

Above-mentioned stringent condition can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and washed film with 0.1 * SSPE in DNA or RNA hybrid experiment.

The recombinant vectors that contains above-mentioned gene also belongs within protection scope of the present invention.

Further, above-mentioned recombinant vectors specifically can be that the dna fragmentation that will contain promotor and above-mentioned gene inserts in the multiple clone site of plasmid pSET152 the recombinant expression vector that obtains; The nucleotide sequence of the described dna fragmentation that contains promotor and above-mentioned gene is shown in sequence in the sequence table 5.

Containing above-mentioned expression of gene box or transgenic cell line also belongs within protection scope of the present invention.

The reorganization bacterium that contains above-mentioned gene also belongs within protection scope of the present invention.

Further, above-mentioned reorganization bacterium is that above-mentioned recombinant vectors is imported the reorganization bacterium that obtains in the purpose Avid kyowamycin.

The above-mentioned purpose Avid kyowamycin can be Avid kyowamycin (Streptomyces avermitilis) ZLX6003CGMCC № .3229.ZLX6003 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and (be called for short CGMCC, the address was: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city) on 08 18th, 2009.ZLX6003 is the Avrmectin superior strain that obtains by the traditional breeding way seed selection, produces industrial being applied to, produces the grey spore on flat board.

Further, above-mentioned reorganization bacterium is Avid kyowamycin (Streptomyces avermitilis) ZLX6056 CGMCC № .3796.ZLX6056 is preserved in Chinese microorganism strain preservation board of trustee reason person on May 4th, 2010 to be understood the common micro-organisms center (be called for short CGMCC, the address is: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), preserving number is CGMCC No.3796.There are 6 mutating acids in sudden change HrdB among the engineering strain ZLX6056, is respectively A137S, K139E, E163G, M356V, V357A, M389I.

ZLX6003 and ZLX6056 all are that aerobic Gram-positive is had a liking for warm actinomycetes, form the aerial hyphae of the tight spiral that divides living mycelia of dendritic base and length.Spore chain is made of 15 or above ganoid circle or avette spore.On oat medium, form gray gas and give birth to spore ball, bacterium colony back side chocolate.On peptone yeast extract and iron substratum, produce melanochrome.

Above-mentioned albumen, above-mentioned gene and the arbitrary above-mentioned application of reorganization bacterium in producing Avrmectin also belong within protection scope of the present invention.

The present invention's RNA polymerase Sigma Factors in confirming Avid kyowamycin increases production Avrmectin to be had on the direct acting basis, by its encoding gene hrdB orthogenesis is made up the mutational vector storehouse, and import in the Avid kyowamycin screening and obtain the recombinant bacterial strain that output improves, wherein synergy gene hrdB is for being fit to the biosynthetic optimization state of Avrmectin.

The following method of concrete employing makes up engineering bacteria, the design primer is by having the hrdB gene of self promotor in the wild bacterium of pcr amplification Avrmectin, structure contains the integrated expression plasmid of hrdB gene, and with the recombinant plasmid transformed that the builds bacterium storehouse that obtains recombinating in the Avid kyowamycin bacterial strain, the method by high flux screening obtains the reorganization bacterium that output improves.The above expression vector is integrated expression vector, and the energy stable application is in production, and the carrier that sets out is intestinal bacteria-streptomycete shuttle vectors pSET152.

The method that hrdB expression vector sudden change storehouse is transformed Avid kyowamycin is preferably the protoplast transformation method that PEG mediates, for improving transformation efficiency, the method that can directly transform by electricity when making up is transformed into mutation expression carrier storehouse among the intestinal bacteria ET12567 of restriction modification effect defective, collects the resistance bacterium colony and therefrom extracts the protoplastis that plasmid transforms Avid kyowamycin again; But also can adopt method commonly used in other bioengineering field, for example electrotransformation, conjugal transfer method etc.

The starting strain that is used to make up the reorganization bacterium can be the bacterial strain of any one Avid kyowamycin, considering superior strain by the more difficult raising output of traditional mutafacient system, is starting strain with existing Avrmectin high yield industrial strain (Avid kyowamycin (Streptomyces avermitilis) ZLX6003 CGMCC 3229) among the present invention.The reorganization bacterium that hrdB gene after the sudden change provided by the invention obtains after importing in the ZLX6003 bacterial strain can reach 5732.05 ± 91.26 μ g/ml in the Avrmectin output of shake-flask culture after 240 hours, is 148.23% of starting strain Avrmectin output.

Engineering bacteria ZLX6056 of the present invention, growth curve on 180 tons of jars shows that this genetic engineering bacterium has kept the good shape of former superior strain, output reaches 6382ug/ml, can well adapt to large scale fermentation, can be directly used in the fermentative production of Avrmectin, the output of Avrmectin is improved, thereby reduce production costs.

Be used for the σ that the present invention relates in the work of the present invention ^HrdBBe the main Sigma Factors in the streptomycete, mainly be responsible for the regulation and control of elementary metabolic gene transcriptional level.The complete sequence of encoding gene hrdB gene and both sides fragment sequence can be retrieved in public database and obtain, be positioned at the base of from 2976855 to 2978393 (on the complementary strands) on the genome, its coded protein sequence is numbered NP_823620 in the Genbank database, GI number is 29606091.Though the concurrent after measured table of Avid kyowamycin genome sequence, the complete sequence of hrdB can obtain in the Genbank database, but, about this gene for the biosynthetic influence of streptomycete secondary metabolite and unclear, in existing literature, also do not utilize this gene to improve the report of yield of streptomycete antibiotic.The present invention has confirmed hrdB by experiment for the biosynthetic regulating and controlling effect of Avrmectin, and utilizes the genetic manipulation of this gene to improve the output of Avrmectin.

Description of drawings

Fig. 1 HrdB albumen vivoexpression and purifying and in-vitro transcription testing and verification its for the biosynthetic regulation and control of Avrmectin; 1A) outer abduction delivering of HrdB proteoplast and purifying, 1B) the in-vitro transcription test confirms that it is for the biosynthetic regulation and control of Avrmectin.

Fig. 2 contains the plasmid map of the recombinant plasmid pZY126 of hrdB gene and himself promotor.

Fig. 3 is the abamectin fermented unit of high yield industry Avid kyowamycin and different transformants thereof.

Fig. 4 analyzes for the mutational site of sudden change Sigma Factors among the Avid kyowamycin sudden change high yield bacterium ZLX6056.

Fig. 5 is the fermentation unit graphic representation of industrial strain ZLX6003 and sudden change high yield bacterium ZLX6056 of setting out.

Embodiment

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

Among the following embodiment, if no special instructions, be ordinary method.

The regulating and controlling effect that embodiment 1, HrdB albumen are transcribed Avrmectin biosynthesizing specificity regulatory gene aveR

One, vivoexpression HrdB albumen

1, the amplification of hrdB structure gene and purifying

The design primer, being used to increase is positioned at hrdB structural gene sequence on the genomic dna, upstream primer HBNde01:5 '-GC CATATGTCGGCCAGCACATCC-3 ', be positioned at hrdB gene start codon ATG position, downstream primer HBSal02:5 '--GCGTCGACTAGTCGAGGTAGTCGC-3 ', be positioned at hrdB gene terminator codon TAG place, the base of band underscore is restriction enzyme NdeI and SalI recognition site, and amplified production should be 1540bp.

(be preserved in CGMCC, genomic dna NO.3229) is a template, is primer with HBNde01 and HBSal02 with Avid kyowamycin ZLX6003, carry out pcr amplification, adopt the LA-Taq enzyme and the GC buffer I of TaKaRa company to carry out pcr amplification, amplification condition is 95 ℃, 3min; (95 ℃, 1min; 60 ℃, 1min; 72 ℃, 1.5min) * 25 circulation; 72 ℃, 10min.Amplified production is carried out agarose gel electrophoresis detect, a specific amplified band is arranged at about 1.5kb place.

2, the HrdB protein expression vector makes up

Reclaim test kit with agarose gel and cut glue recovery pcr amplification product, be connected with T carrier pMD 18-T (TaKaRa company), show through the order-checking comparison, the fragment that is increased is hrdB gene structure gene (encoding sequence shown in sequence in the sequence table 1, the albumen shown in the sequence 2 in the codified sequence table) really.The T carrier that will contain hrdB structure gene reclaims the hrdB fragment of 1.5kb after NdeI and SalI enzyme are cut, with this fragment with link to each other the competent cell of connection product transformed into escherichia coli DH5a with the carrier pET28b (Novagen) that the XhoI enzyme is cut through NdeI.From transformant, extract plasmid and carry out enzyme and cut checking, with correct recombinant plasmid called after pZY165 respectively.

Two, protein expression purifying and HrdB albumen are to the influence of transcribing of aveR

1, HrdB protein expression and purifying

Choose mono-clonal from flat board and (contain corresponding microbiotic) to the LB liquid nutrient medium of 5ml, incubated overnight is to logarithmic phase; Adding IPTG is that 0.4mM continues to cultivate 10h to final concentration, gets 200ul bacterium liquid and carries out SDS-PAGE gel electrophoresis detection protein expression situation.Behind ultrasonication cell under the non-sex change condition, carry out preliminary purification with the HisTrap HP affinity column (GE Healthcare) of 5ml after, suction filtration carries out molecular sieving after concentrating.Albumen behind the purifying (HrdB albumen) result is shown in Figure 1A.

2, HrdB albumen is to the specificity regulating and controlling effect of aveR

RNA center enzyme and the quantitative HrdB albumen of purifying are mixed by 1: 1 concentration ratio, and the polysaccharase mixture is placed on 30 ℃ of water-bath 5min; Add 3.5ul and contain [a-32P] CTP (400Ci/mmol) substrate mixture continuation temperature bath 15 minutes; Reflection finishes back adding sample-loading buffer and carries out 5%polyacrylamide gel (containing 7M urea) electrophoresis detection, and the result is shown in Figure 1B after the radioautograph.

Fig. 2 result shows that HrdB albumen can be in external specific identification aveR upstream promoter sequence, and startup HrdB albumen transcribing to aveR, and increase along with the HrdB protein concentration, the amount of the transcription product of aveR presents and increases progressively trend, and the result shows that HrdB albumen can participate in the transcriptional level of aveR.

In-vitro transcription test method reference literature (Hahn MY, Bae JB, Park JH, Roe JH.Isolation andcharacterization of Streptomyces coelicolor RNA polymerase, its sigma, and antisigmafactors.Methods Enzymol 2003,370:73-82).

The structure of embodiment 2, hrdB expression vector

One, the construction of recombinant plasmid that contains hrdB gene and himself promotor

1, contains the amplification of the hrdB gene of self promotor

The design primer is used for pcr amplification and is positioned at the hrdB gene [NC_003155.4, complement (2976855..2978393)] that contains self promotor on the Avid kyowamycin karyomit(e), upstream primer PodP1:5 '-CAC TCTAGACCCTGAGGTGGAGCGTGTG-3 ' is positioned at hrdB upstream from start codon 649bp place, downstream primer PodP2:5 '-CTC GAATTCGGTCATGGAATACCCAGAGTGAT-3 ' is positioned at 120bp place, hrdB terminator codon downstream, and the base of band underscore is restriction enzyme XbaI and EcoRI recognition site, and amplified production should be 2352bp.

Total DNA with Avid kyowamycin wild type strain ATCC31267 is a template, is primer with PodP1 and PodP2, adopts the LA-Taq enzyme and the GC buffer I of TaKaRa company to carry out pcr amplification, and amplification condition is 95 ℃, 3min; (95 ℃, 1min; 64.6 ℃, 1min; 72 ℃, 2.5min) * 25 circulation; 72 ℃, 10min.Amplified production is carried out agarose gel electrophoresis detect, a specific amplified band is arranged at about 2.3kb place.

2, the structure of recombinant plasmid pZY126

Reclaim test kit with agarose gel and cut glue and reclaim pcr amplification product, be connected with T carrier pMD 18-T (TaKaRa company), show through the order-checking comparison, the fragment that is increased is really for containing the hrdB gene of self promotor.The T carrier that will contain the hrdB gene is after XbaI and EcoRI enzyme are cut, reclaim the hrdB fragment of 2.3kb, with this fragment and carrier pSET152 (the Bierman M that cuts through same enzyme, Logan R, O ' Brien K, et al.Plasmid cloningvectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.Gene, 1992,116:43-49) link to each other, connect the competent cell of product transformed into escherichia coli DH5 α.From transformant, extract plasmid and carry out enzyme and cut checking, with correct recombinant plasmid called after pZY126 respectively.The plasmid map of pZY126 as shown in Figure 2.

Two, based on the structure in the hrdB transgenation storehouse of pZY126 plasmid

1, the amplification and the purifying of sudden change hrdB structure gene

The design primer, be used to increase and be positioned at hrdB structural gene sequence on the pZY126 plasmid, upstream primer PodP3:5 '-TGTTCGTGTCGGCCAGCAC-3 ', be positioned at 5bp place, hrdB gene start codon upstream, downstream primer PodP4:5 '-TGCGTACAGCCGAGACCTAGTC-3 ', be positioned at 14bp place, hrdB gene terminator codon downstream, amplified production should be 1560bp.

With the pZY126 plasmid DNA behind the purifying is template, is primer with PodP3 and PodP4, carries out pcr amplification, adopts the Mutazyme II DNA Polymerase enzyme of Stratagene company to carry out pcr amplification, and amplification condition is 95 ℃, 2min; (95 ℃, 1min; 59 ℃, 1min; 72 ℃, 1.5min) * 30 circulation; 72 ℃, 10min.Amplified production is carried out agarose gel electrophoresis detect, a specific amplified band is arranged at about 1.6kb place.

Adopt PCR product purification test kit purifying to reclaim pcr amplification product, get the 1ul purified product and carry out agarose gel electrophoresis and carry out the quantitative of nanodrop detection carrying out purified product.

2, the structure that contains the segmental pZY126* plasmid of sudden change hrdB sheet phase library

With PCR product behind the above-mentioned purifying is primer, and according to preparing reaction system in the GeneMorph II EZClone product description, amplification condition is 95 ℃, 1min; (95 ℃, 50sec; 60 ℃, 50sec; 68 ℃, 3min) * 25 circulation; Reaction was placed 2 minutes after finishing on ice.

In amplified production, add the DpnI enzyme, be used for the plasmid DNA of template with digest amplification product Central Plains.

So far, containing the segmental pZY126* expression plasmid of sudden change hrdB carrier storehouse structure finishes.

The conversion in embodiment 3, mutant plasmid storehouse

One, the amplification in mutant plasmid storehouse

Owing to there is very strong restriction modification effect in the Avid kyowamycin, directly transform Avid kyowamycin with extraction from the plasmid of E.coli DH5 α, transformation efficiency is extremely low, sometimes even can not get transformant.And use plasmid from the recipient bacterium E.coli ET12567 of modification without limits, its transformation efficiency obviously improves.Therefore, the mutant plasmid storehouse that builds and control plasmid are transformed into E.coli ET12567 (pUZ8002) respectively earlier, and (non-patent literature of putting down in writing this material is MacNeil DJ, Gewain KM, Ruby CL, et al (1992) Analysis of Streptomyces avermitilisgenes required for avermectin biosynthesis utilizing a novel integration vector.Gene1992,111:61-68., the public can obtain from Institute of Microorganism, Academia Sinica) obtaining non-methylated DNA, and then transform the protoplastis of Avid kyowamycin with non-methylated plasmid DNA.

To make up the competent cell that contains sudden change hrdB segmental pZY126* expression plasmid carrier storehouse and original plasmid pZY126 in contrast and pSET152 electric shock Transformed E .coli ET12567 by embodiment 1, coat and contain card and receive the LB flat board of mycin, paraxin and apramycin, 37 degree are placed and are cultivated.Treat to grow on the flat board visible bacterium colony, collect all bacterium colonies with fresh liquid LB after, add 15% glycerine and be saved in-80 ℃, or directly insert to contain and cultivate further amplification plasmid among the corresponding antibiotic LB, extract plasmid DNA, enzyme is cut checking.

Two, the mutant plasmid storehouse transforms Avid kyowamycin

This example has selected for use Avid kyowamycin (Streptomyces avermitilis) ZLX6003 CGMCC 3229 as starting strain, ZLX6003 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and (be called for short CGMCC, the address was: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city) on 08 18th, 2009.ZLX6003 is the Avrmectin superior strain that obtains by the traditional breeding way seed selection, produces industrial being applied to, produces the grey spore on flat board.

The protoplastis of preparation Avid kyowamycin bacterial strain ZLX6003, transform the protoplastis of starting strain ZLX6003 with each plasmid (pZY126*, pZY126 and pSET152) that from E.coliET12567 (pUZ8002), extracts in the step 1, be applied on RM 14 flat boards of the not added with antibiotic that has dried up, behind 28 ℃ of cultivation 16-20h, on flat board, cover the aqueous solution that 1mL contains the 1mg apramycin, continue to cultivate 7-10 days at 28 ℃, the bacterium colony that grows is transformant.The uniform transformant of picking colony form is inoculated on the MS flat board that contains the 20ug/mL apramycin at random, cultivates for 28 ℃ and recovers to produce robe in 7 days.Transformant carries out next step fermentation research after plasmid extraction and PCR checking correctly.

The preparation of colibacillary conversion in the present embodiment, Avid kyowamycin protoplastis and method for transformation, RM 14 and MS culture medium preparation referring to the Ph D dissertation of Zhang Xiaolin (Zhang Xiaolin. the genetic modification of polyketide synthase gene in the Avid kyowamycin. the doctorate paper, 2004, Beijing: China Agricultural University).

The fermentation of embodiment 4, transformant

One, the fermentation of ZLX6003 and transformant thereof and volume analysis

1, the high flux screening of Avid kyowamycin

Avid kyowamycin starting strain ZLX6003 and different transformant thereof are after growing abundant spore on the slant medium, Avrmectin yield level to the Avid kyowamycin transformant is tested, adopt high-throughput screening method to carry out primary dcreening operation (Gao H, Liu M, Zhou X et al.Identification of avermectin-high-producing strains byhigh-throughput screening methods.Appl Microbiol Biotechnol, 2009,85:1219-1225.)

The bacterial strain that the output that primary dcreening operation obtains improves is elected to be shake flask fermentation and verifies, takes inclined-plane lawn 1cm ², the went out seed bottle of seed culture medium of bacterium of 40mL is equipped with in its access, 28 ℃ of shaking tables were cultivated 44-48 hour, rotating speed 200rpm, rotation radius is 50mm, obtains seed culture fluid.Getting above-mentioned seed culture fluid is inoculated in by the inoculum size of 5% (volume percent) and 30mL is housed went out in the triangular flask of fermention medium of bacterium, 28 ℃ of shaking tables were cultivated 10 days, put bottle, with the fermentation unit of HPLC method mensuration Avrmectin, it is described that concrete HPLC is analyzed as follows the step of stating 2.

Slant medium in the present embodiment, the preparation of seed culture medium and fermention medium is referring to existing patent (No.200810227639.8, a kind of method and special strain therefore thereof for preparing Avrmectin).

2, the HPLC of tunning analyzes

1) sample preparation: get the 1.0mL fermented liquid, add 9.0mL methyl alcohol, with speed (rotation radius the is 50mm) jolting of 200rpm 6 hours, with the centrifugal 5min of the speed of 8000rpm;

2) get the 1mL supernatant,, obtain filtrate and carry out the HPLC analysis with the filtering with microporous membrane of 0.45um.

3) get the ferment filtrate of step 1, automatic sampler sample introduction, sample size 10ul;

With methyl alcohol: water (volume ratio is 9: 1) is that moving phase is separated, and flow velocity is 1mL/min, utilizes the UV detector to detect at wavelength 245nm place and forms separating spectrum automatically; Under this chromatographic condition, (DR, retention time Germany) is about 6 minutes to Avrmectin B1a standard substance.Retention time is the elution peak area of locating about 6 minutes in the calculating ferment filtrate, calculates the amount of Avrmectin B1a.3 repetitions are established in experiment, and the result takes the mean.

The fermentation result of Fig. 3 shows that the output of Avrmectin output behind the conversion starting strain ZLX6003 of control plasmid pZY126 (representing with pSET152-hrdB among the figure) and blank plasmid pSET152 and the Avrmectin of starting strain ZLX6003 does not have significant difference; And the output of the more former starting strain ZLX6003 of output of the Avrmectin of the bacterial strain that above-mentioned output improves (transformant that contains sudden change hrdB gene) has increased by 52%, this superior strain is as engineering bacteria, be Avid kyowamycin (Streptomyces avermitilis) ZLX6056 CGMCC № .3796, be preserved in Chinese microorganism strain preservation board of trustee reason person on May 4th, 2010 and understand the common micro-organisms center, preserving number is CGMCCNo.3796.

Two, sudden change hrdB gene sequencing in the recombinant bacterial strain and sequential analysis and to the checking of Avrmectin yield effect

1, the order-checking and the sequential analysis of sudden change hrdB gene

Extract the genomic dna of engineering bacteria ZLX6056, carry out complete degestion with PstI, and behind ethanol sedimentation purifying endonuclease bamhi, connect certainly, will connect product transformed into escherichia coli competent cell certainly, be applied to the LB flat board that contains the apramycin resistance, 37 ℃ are cultured to and white colony occurs.Picking list bacterium colony is cultivated the back and is extracted plasmid DNA, that reclaims 2.3kb after XbaI and EcoRI enzyme are cut contains promotor in interior sudden change hrdB gene fragment, with the gene fragment of this 2.3kb and transformed into escherichia coli DH5 α competent cell after the carrier pUC18 that cuts through same enzyme is connected, be applied to the LB flat board that contains amicillin resistance, 37 ℃ are cultured to and white colony occurs.Picking list bacterium colony is cultivated the back and is extracted plasmid DNA, send Beijing China major company to check order after enzyme is cut checking.Sequencing result shows that the nucleotide sequence of the gene fragment of this 2.3kb is shown in sequence in the sequence table 5.Wherein the nucleotide sequence from shown in the 658-2197 position of 5 ' end of sequence 5 is consistent with sequence 3 in the sequence table, is the sequence of the hrdB gene after the sudden change, the hrdB albumen in the codified sequence table after the sudden change shown in the sequence 4.

Carry out that sequence is compared and to analyzing its conservative functional domain at the HrdB albumen after the sudden change shown in the sequence 4 and the HrdB albumen before the sudden change shown in the sequence 2.The result as shown in Figure 4, the sudden change before and after protein sequence, total length is 512AA.Concentrate on 1.1 and 2.4 districts in the mutational site.There are 6 point mutation in the hrdB albumen after sudden change, are respectively A137S, K139E, E163G, M356V, V357A, and M389I.Bibliographical information 2.4 districts mainly are responsible for the combination in promotor-10 district of gene, and stability is very high, and have 2 point mutation in engineering bacteria ZLX6056.The result further shows, increases the sudden change hrdB gene of a copy among the engineering bacteria ZLX6056, has influenced the gene expression dose of Avrmectin biosynthesizing relational approach, thereby has made Avrmectin output be improved.

2, sudden change hrdB fragment is to the checking of Avrmectin yield effect

1) structure of recombinant expression vector

(non-patent literature of putting down in writing this material is Bierman M at interior sudden change hrdB gene fragment and the carrier pSET152 behind XbaI, EcoRI double digestion with the promotor that contains of the 2.3kb that reclaims in the step 1, Logan R, O ' BrienK, et al.Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli toStreptomyces spp.Gene, 1992,116:43-49), the public can obtain from institute of microbiology of the Chinese Academy of Sciences) connect, obtain recombinant expression vector.(non-patent literature of putting down in writing this material is MacNeil DJ with this recombinant expression vector transformed into escherichia coli E.coli ET12567 then, Gewain KM, Ruby CL, et al (1992) Analysis ofStreptomyces avermitilis genes required for avermectin biosynthesis utilizing a novelintegration vector.Gene 111:61-68., the public can obtain from institute of microbiology of the Chinese Academy of Sciences) competent cell, be applied to and contain paraxin, the LB flat board of kantlex and apramycin resistance, 37 ℃ are cultured to and white colony occurs.Picking list bacterium colony is cultivated the back and is extracted plasmid DNA, cuts the laggard pacing preface of checking through enzyme, shows that the recombinant expression vector that obtains is that sequence 5 described dna fragmentations are inserted between the XbaI of pSET152 and the EcoRI restriction enzyme site and constitute in the sequence table.

2) transform Avid kyowamycin ZLX6003 bacterial strain and fermenting

The recombinant expression vector of step 1) acquisition is transformed into the protoplastis of Avid kyowamycin ZLX6003 bacterial strain, be applied on RM 14 flat boards of the not added with antibiotic that has dried up, behind 28 ℃ of cultivation 16-20h, on flat board, cover the aqueous solution that 1mL contains the 1mg apramycin, continue to cultivate 7-10 days at 28 ℃, the bacterium colony that grows is transformant.The uniform transformant of picking colony form is inoculated on the MS flat board that contains the 20ug/mL apramycin at random, cultivates for 28 ℃ and recovers to produce robe in 7 days.。The transformant called after pZY148-hrdB that the PCR checking is correct ^ZLX6056, the transformant verified of 5 PCR of random choose then, the method for step 1 step 2 is fermented in embodiment 4.

Simultaneously carrier pSET152 is transformed into the protoplastis of Avid kyowamycin ZLX6003 bacterial strain, obtains the empty carrier control strain.

Then empty carrier control strain, starting strain ZLX6003 and engineering bacteria ZLX6056 are carried out above-mentioned fermentation culture equally.

Fermenting experiment repeats 3 times, and the result is shown in table 2 (being decided to be 100% with the B1a output of starting strain ZLX6003 fermentation after 240 hours), and bacterium pZY148-hrdB recombinates ^ZLX6056Ferment and to reach 148.23% after 240 hours, can recover growth and the output characteristic of engineering bacteria ZLX6056, and the B1a output of the output of empty carrier control strain and starting strain ZLX6003 does not have significant difference.

Can determine that thus the hrdB gene after the sudden change shown in the sequence 3 can improve Avrmectin output in the sequence table.

The functional verification of the hrdB gene after table 2, the sudden change

Embodiment 5, engineering strain genetic stability detect

Engineering strain ZLX6056 of the present invention is carried out the inclined-plane to go down to posterity, passed for 5 generations altogether, 28 ℃ are cultured to and grow (about 12～15 days) behind the vigorous spore, whenever the foster bacterium that obtains of being commissioned to train is all fermented according to the shake flask fermentation method in the embodiment 4 step 1 steps 1, ferment and extract by method described in the embodiment 4 step 1 steps 2, and calculate the ability that bacterial strain is produced Avrmectin B1a.3 repetitions are established in experiment, and the result takes the mean.The result shows engineering strain ZLX6056 of the present invention after going down to posterity for 5 times, and throughput can also keep original level, shows that the genetic stability of engineering strain ZLX6056 of the present invention is good.

The fermentation of embodiment 6, engineering strain is amplified

The Avrmectin of engineering strain ZLX6056 on 180 tons of fermentor tanks of the hrdB gene after present embodiment has compared starting strain ZLX6003 and contained sudden change produces plain ability, and the sampling of fermenting process discontinuous is analyzed the changing conditions of Avrmectin output.

Wherein fermentation step is as follows:

In the pond that feeds intake, by the formulated fermention medium and transfer pH, prepared culture medium is pumped into 180m ³Fermentor tank in, behind the polyphony pH, finish postcooling to 28 degree of 121 ℃～130 ℃ sterilizations 30 minutes～1 hour, sterilization, standby behind the polyphony pH.To be cultured to the seed of logarithmic phase, under aseptic situation with 10m ³(± 20%) left and right sides seed liquor pumps in the fermentor tank of the above-mentioned bacterium of going out, starts the fermentation culture program, 28～29 ℃ of cultivations, DO is about 50% in control, and pH is controlled at about 7.8, and stirring velocity is 0～100rpm in earlier stage, 24h is later on 100～200rpm, is about 200～300rpm after 72 hours.Regularly detect mycelial growth and produce plain situation, fermentor tank is put jar after moving about 312 hours.

The preparation of seed culture medium and fermention medium is referring to existing patent (No.200810227639.8, a kind of method and special strain therefore thereof for preparing Avrmectin) in the present embodiment.

The result as shown in Figure 5, engineering strain ZLX6056 is in the advantage that still can keep increase of production on the large scale fermentation jar after 200 hours, the ability of engineering strain production Avrmectin B1a of the present invention reaches 6382 μ g/ml fermented liquids in 312 hours, improves 53.1% than the plain ability of the product of starting strain ZLX6003 bacterial strain (4167 μ g/ml).

Sequence table

＜110〉Institute of Microorganism, Academia Sinica

＜120〉a kind of gene engineering method and special strain therefore thereof of producing Avrmectin

<130>CGGNARL102312

<160>5

<210>1

<211>1539

<212>DNA

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>1

gtgtcggcca gcacatcccg tacgctcccg ccggagatcg ccgagtccgt ctctgtcatg 60

gcgctcatcg agcggggaaa ggctgagggg cagatcgccg gcgatgacgt gcgtcgggcc 120

ttcgaagctg accagattcc ggccactcag tggaagaacg tactgcgcag cctcaaccag 180

atcctcgagg aagagggtgt gacgctgatg gtcagtgccg cggagcccaa gcgcacccga 240

aagagcgtcg cagcgaagag tccggccaag cgcaccgcca ccaagaccgt cgcggcgaag 300

acggtgactg ccaagaaggc gaccgccacc gccgccccgg ctgtgcccgt cggcgacgat 360

ccggctgagg acgcgtccgc caagaaggca gctgccaaga agacgaccgc caagaaggcg 420

gtcgcgaaga agaccgtcgc caagaagacg gcggccaaga agaccaccgg caagaaggac 480

gacgtcgagc tgctcgacga cgaggcggtc gaggagaccg ctgcacccgg caaggccggc 540

gaggagcccg agggcaccga gaacgccggc ttcgtactct ccgacgagga cgaggacgac 600

gcgcccgcgc agcaggtcgc cgcggccggt gccaccgccg acccggtcaa ggactacctc 660

aagcagatcg gcaaggtccc cctgctcaac gccgagcagg aggtcgagct cgccaagcgc 720

atcgaggcgg gcctcttcgc cgaggacaag ctggccaacg ccgacaagct tgcccccaag 780

ctcaagcgcg agctggagat catcgccgag gacggccgcc gcgccaagaa ccacctcctg 840

gaggccaacc tccgtctggt ggtctccctg gccaagcgct acaccggccg cggcatgctc 900

ttcctggacc tcatccagga gggcaacctc ggtctgatcc gcgcggtgga gaagttcgac 960

tacaccaagg gctacaagtt ctccacgtac gccacctggt ggatccgtca ggcgatcacc 1020

cgcgccatgg ccgaccaggc ccgcaccatc cgtatcccgg tgcacatggt cgaggtcatc 1080

aacaagctcg cgcgcgtgca gcgtcagatg ctccaggacc tgggccgtga gcccaccccg 1140

gaggagctgg ccaaggagct cgacatgacc cctgagaagg tcatcgaggt ccagaagtac 1200

ggccgtgagc ccatctcgct gcacaccccg ctgggtgagg acggtgacag cgagttcggt 1260

gacctcatcg aggactccga ggccgtcgtc ccggccgacg cggtcagctt cacgctcctc 1320

caggagcagc tgcactctgt cctcgacacc ctgtcggagc gcgaggcggg cgtcgtctcg 1380

atgcgcttcg gtctcaccga cggtcagccg aagactctcg acgagatcgg caaggtgtac 1440

ggcgtgacgc gtgagcgcat ccgccagatc gagtccaaga cgatgtcgaa gctgcgtcac 1500

ccgtcgcgtt cgcaggtgct gcgcgactac ctcgactag 1539

<210>2

<211>512

<212>PRT

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>2

Val Ser Ala Ser Thr Ser Arg Thr Leu Pro Pro Glu Ile Ala Glu Ser

1 5 10 15

Val Ser Val Met Ala Leu Ile Glu Arg Gly Lys Ala Glu Gly Gln Ile

20 25 30

Ala Gly Asp Asp Val Arg Arg Ala Phe Glu Ala Asp Gln Ile Pro Ala

35 40 45

Thr Gln Trp Lys Asn Val Leu Arg Ser Leu Asn Gln Ile Leu Glu Glu

50 55 60

Glu Gly Val Thr Leu Met Val Ser Ala Ala Glu Pro Lys Arg Thr Arg

65 70 75 80

Lys Ser Val Ala Ala Lys Ser Pro Ala Lys Arg Thr Ala Thr Lys Thr

85 90 95

Val Ala Ala Lys Thr Val Thr Ala Lys Lys Ala Thr Ala Thr Ala Ala

100 105 110

Pro Ala Val Pro Val Gly Asp Asp Pro Ala Glu Asp Ala Ser Ala Lys

115 120 125

Lys Ala Ala Ala Lys Lys Thr Thr Ala Lys Lys Ala Val Ala Lys Lys

130 135 140

Thr Val Ala Lys Lys Thr Ala Ala Lys Lys Thr Thr Gly Lys Lys Asp

145 150 155 160

Asp Val Glu Leu Leu Asp Asp Glu Ala Val Glu Glu Thr Ala Ala Pro

165 170 175

Gly Lys Ala Gly Glu Glu Pro Glu Gly Thr Glu Asn Ala Gly Phe Val

180 185 190

Leu Ser Asp Glu Asp Glu Asp Asp Ala Pro Ala Gln Gln Val Ala Ala

195 200 205

Ala Gly Ala Thr Ala Asp Pro Val Lys Asp Tyr Leu Lys Gln Ile Gly

210 215 220

Lys Val Pro Leu Leu Asn Ala Glu Gln Glu Val Glu Leu Ala Lys Arg

225 230 235 240

Ile Glu Ala Gly Leu Phe Ala Glu Asp Lys Leu Ala Asn Ala Asp Lys

245 250 255

Leu Ala Pro Lys Leu Lys Arg Glu Leu Glu Ile Ile Ala Glu Asp Gly

260 265 270

Arg Arg Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu Val Val

275 280 285

Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Leu Phe Leu Asp Leu

290 295 300

Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys Phe Asp

305 310 315 320

Tyr Thr Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp Ile Arg

325 330 335

Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile Arg Ile

340 345 350

Pro Val His Met Val Glu Val Ile Asn Lys Leu Ala Arg Val Gln Arg

355 360 365

Gln Met Leu Gln Asp Leu Gly Arg Glu Pro Thr Pro Glu Glu Leu Ala

370 375 380

Lys Glu Leu Asp Met Thr Pro Glu Lys Val Ile Glu Val Gln Lys Tyr

385 390 395 400

Gly Arg Glu Pro Ile Ser Leu His Thr Pro Leu Gly Glu Asp Gly Asp

405 410 415

Ser Glu Phe Gly Asp Leu Ile Glu Asp Ser Glu Ala Val Val Pro Ala

420 425 430

Asp Ala Val Ser Phe Thr Leu Leu Gln Glu Gln Leu His Ser Val Leu

435 440 445

Asp Thr Leu Ser Glu Arg Glu Ala Gly Val Val Ser Met Arg Phe Gly

450 455 460

Leu Thr Asp Gly Gln Pro Lys Thr Leu Asp Glu Ile Gly Lys Val Tyr

465 470 475 480

Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr Met Ser

485 490 495

Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr Leu Asp

500 505 510

<210>3

<211>1539

<212>DNA

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>3

gtgtcggcca gcacatcccg tacgctcccg ccggagatcg ccgagtccgt ctctgtcatg 60

gcgctcatcg agcggggaaa ggctgagggg cagatcgccg gcgatgacgt gcgtcgggcc 120

ttcgaagctg accagattcc ggccactcag tggaagaacg tactgcgcag cctcaaccag 180

atcctcgagg aagagggtgt gacgctgatg gtcagtgccg cggagcccaa gcgcacccga 240

aagagcgtcg cagcgaagag tccggccaag cgcaccgcca ccaagaccgt cgcggcgaag 300

acggtgactg ccaagaaggc gaccgccacc gccgccccgg ctgtgcccgt cggcgacgat 360

ccggctgagg acgcgtccgc caagaaggca gctgccaaga agacgacctc caaggaggcg 420

gtcgcgaaga agaccgtcgc caagaagacg gcggccaaga agaccaccgg caagaaggac 480

gacgtcgggc tgctcgacga cgaggcggtc gaggagaccg ctgcacccgg caaggccggc 540

gaggagcccg agggcaccga gaacgccggc ttcgtactct ccgacgagga cgaggacgac 600

gcgcccgcgc agcaggtcgc cgcggccggt gccaccgccg acccggtcaa ggactacctc 660

aagcagatcg gcaaggtccc cctgctcaac gccgagcagg aggtcgagct cgccaagcgc 720

atcgaggcgg gcctcttcgc cgaggacaag ctggccaacg ccgacaagct tgcccccaag 780

ctcaagcgcg agctggagat catcgccgag gacggccgcc gcgccaagaa ccacctcctg 840

gaggccaacc tccgtctggt ggtctccctg gccaagcgct acaccggccg cggcatgctc 900

ttcctggacc tcatccagga gggcaacctc ggtctgatcc gcgcggtgga gaagttcgac 960

tacaccaagg gctacaagtt ctccacgtac gccacctggt ggatccgtca ggcgatcacc 1020

cgcgccatgg ccgaccaggc ccgcaccatc cgtatcccgg tgcacgtggc cgaggtcatc 1080

aacaagctcg cgcgcgtgca gcgtcagatg ctccaggacc tgggccgcga gcccaccccg 1140

gaggagctgg ccaaggagct cgacattacc cctgagaagg tcatcgaggt ccagaagtac 1200

ggccgtgagc ccatctcgct gcacaccccg ctgggtgagg acggtgacag cgagttcggt 1260

gacctcatcg aggactccga ggccgtcgtc ccggccgacg cggtcagctt cacgctcctc 1320

caggagcagc tgcactctgt cctcgacacc ctgtcggagc gcgaggcggg cgtcgtctcg 1380

atgcgcttcg gtctcaccga cggtcagccg aagactctcg acgagatcgg caaggtgtac 1440

ggcgtgacgc gtgagcgcat ccgccagatc gagtccaaga cgatgtcgaa gctgcgtcac 1500

ccgtcgcgtt cgcaggtgct gcgcgactac ctcgactag 1539

<210>4

<211>512

<212>PRT

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>4

Val Ser Ala Ser Thr Ser Arg Thr Leu Pro Pro Glu Ile Ala Glu Ser

1 5 10 15

Val Ser Val Met Ala Leu Ile Glu Arg Gly Lys Ala Glu Gly Gln Ile

20 25 30

Ala Gly Asp Asp Val Arg Arg Ala Phe Glu Ala Asp Gln Ile Pro Ala

35 40 45

Thr Gln Trp Lys Asn Val Leu Arg Ser Leu Asn Gln Ile Leu Glu Glu

50 55 60

Glu Gly Val Thr Leu Met Val Ser Ala Ala Glu Pro Lys Arg Thr Arg

65 70 75 80

Lys Ser Val Ala Ala Lys Ser Pro Ala Lys Arg Thr Ala Thr Lys Thr

85 90 95

Val Ala Ala Lys Thr Val Thr Ala Lys Lys Ala Thr Ala Thr Ala Ala

100 105 110

Pro Ala Val Pro Val Gly Asp Asp Pro Ala Glu Asp Ala Ser Ala Lys

115 120 125

Lys Ala Ala Ala Lys Lys Thr Thr Ser Lys Glu Ala Val Ala Lys Lys

130 135 140

Thr Val Ala Lys Lys Thr Ala Ala Lys Lys Thr Thr Gly Lys Lys Asp

145 150 155 160

Asp Val Gly Leu Leu Asp Asp Glu Ala Val Glu Glu Thr Ala Ala Pro

165 170 175

Gly Lys Ala Gly Glu Glu Pro Glu Gly Thr Glu Asn Ala Gly Phe Val

180 185 190

Leu Ser Asp Glu Asp Glu Asp Asp Ala Pro Ala Gln Gln Val Ala Ala

195 200 205

Ala Gly Ala Thr Ala Asp Pro Val Lys Asp Tyr Leu Lys Gln Ile Gly

210 215 220

Lys Val Pro Leu Leu Asn Ala Glu Gln Glu Val Glu Leu Ala Lys Arg

225 230 235 240

Ile Glu Ala Gly Leu Phe Ala Glu Asp Lys Leu Ala Asn Ala Asp Lys

245 250 255

Leu Ala Pro Lys Leu Lys Arg Glu Leu Glu Ile Ile Ala Glu Asp Gly

260 265 270

Arg Arg Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu Val Val

275 280 285

Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Leu Phe Leu Asp Leu

290 295 300

Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys Phe Asp

305 310 315 320

Tyr Thr Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp Ile Arg

325 330 335

Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile Arg Ile

340 345 350

Pro Val His Val Ala Glu Val Ile Asn Lys Leu Ala Arg Val Gln Arg

355 360 365

Gln Met Leu Gln Asp Leu Gly Arg Glu Pro Thr Pro Glu Glu Leu Ala

370 375 380

Lys Glu Leu Asp Ile Thr Pro Glu Lys Val Ile Glu Val Gln Lys Tyr

385 390 395 400

Gly Arg Glu Pro Ile Ser Leu His Thr Pro Leu Gly Glu Asp Gly Asp

405 410 415

Ser Glu Phe Gly Asp Leu Ile Glu Asp Ser Glu Ala Val Val Pro Ala

420 425 430

Asp Ala Val Ser Phe Thr Leu Leu Gln Glu Gln Leu His Ser Val Leu

435 440 445

Asp Thr Leu Ser Glu Arg Glu Ala Gly Val Val Ser Met Arg Phe Gly

450 455 460

Leu Thr Asp Gly Gln Pro Lys Thr Leu Asp Glu Ile Gly Lys Val Tyr

465 470 475 480

Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr Met Ser

485 490 495

Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr Leu Asp

500 505 510

<210>5

<211>2352

<212>DNA

＜213〉Avid kyowamycin (Streptomyces avermitilis)

<400>5

cactctagac cctgaggtgg agcgtgtggt gcccgcgccg cgcgagcact gacggtgcgc 60

cgtgggcggc gcggtcgggg gccgaccgcc ctcgcccgct ctcgcccggc cggtgggccc 120

gacagcgccc gcacggcgaa ccgtctgtca ccggaccccg tgcacgtgtc gccgggctcc 180

gtcggaccct cctgggaccg acggggttcg acggcacgtc ttccgggacc ggcgcggttc 240

gacggcatgc ggagtccggg aatcggcatg gctcggcggc gtacggagcc cgggagccgc 300

tgaggtccga cggcgagcga cccggcggcc aaccgctgat tcggcggccc ggaagtccac 360

cgaccctcgg atcgtgcggc cgcagcggcc atcgttgacc acctatgacc gcatctagtc 420

gtttttgagt ggttacgggg tgtgactcgg gccacgcgga ttgggcgtaa cgctcctcgg 480

cactgcgcga tgacctaaga ggtgacagcc gaggagggaa tacggacgcc gtttacggcg 540

ctgtgcatct tcccggcccc acccgcgccg tcggcccatc cccaagtcgg cggtcgtcgg 600

ttcctgtccg ttacggacgg ggccggaagc cgttttccaa cgttccgaga ggttgttcgt 660

gtcggccagc acatcccgta cgctcccgcc ggagatcgcc gagtccgtct ctgtcatggc 720

gctcatcgag cggggaaagg ctgaggggca gatcgccggc gatgacgtgc gtcgggcctt 780

cgaagctgac cagattccgg ccactcagtg gaagaacgta ctgcgcagcc tcaaccagat 840

cctcgaggaa gagggtgtga cgctgatggt cagtgccgcg gagcccaagc gcacccgaaa 900

gagcgtcgca gcgaagagtc cggccaagcg caccgccacc aagaccgtcg cggcgaagac 960

ggtgactgcc aagaaggcga ccgccaccgc cgccccggct gtgcccgtcg gcgacgatcc 1020

ggctgaggac gcgtccgcca agaaggcagc tgccaagaag acgacctcca aggaggcggt 1080

cgcgaagaag accgtcgcca agaagacggc ggccaagaag accaccggca agaaggacga 1140

cgtcgggctg ctcgacgacg aggcggtcga ggagaccgct gcacccggca aggccggcga 1200

ggagcccgag ggcaccgaga acgccggctt cgtactctcc gacgaggacg aggacgacgc 1260

gcccgcgcag caggtcgccg cggccggtgc caccgccgac ccggtcaagg actacctcaa 1320

gcagatcggc aaggtccccc tgctcaacgc cgagcaggag gtcgagctcg ccaagcgcat 1380

cgaggcgggc ctcttcgccg aggacaagct ggccaacgcc gacaagcttg cccccaagct 1440

caagcgcgag ctggagatca tcgccgagga cggccgccgc gccaagaacc acctcctgga 1500

ggccaacctc cgtctggtgg tctccctggc caagcgctac accggccgcg gcatgctctt 1560

cctggacctc atccaggagg gcaacctcgg tctgatccgc gcggtggaga agttcgacta 1620

caccaagggc tacaagttct ccacgtacgc cacctggtgg atccgtcagg cgatcacccg 1680

cgccatggcc gaccaggccc gcaccatccg tatcccggtg cacgtggccg aggtcatcaa 1740

caagctcgcg cgcgtgcagc gtcagatgct ccaggacctg ggccgcgagc ccaccccgga 1800

ggagctggcc aaggagctcg acattacccc tgagaaggtc atcgaggtcc agaagtacgg 1860

ccgtgagccc atctcgctgc acaccccgct gggtgaggac ggtgacagcg agttcggtga 1920

cctcatcgag gactccgagg ccgtcgtccc ggccgacgcg gtcagcttca cgctcctcca 1980

ggagcagctg cactctgtcc tcgacaccct gtcggagcgc gaggcgggcg tcgtctcgat 2040

gcgcttcggt ctcaccgacg gtcagccgaa gactctcgac gagatcggca aggtgtacgg 2100

cgtgacgcgt gagcgcatcc gccagatcga gtccaagacg atgtcgaagc tgcgtcaccc 2160

gtcgcgttcg caggtgctgc gcgactacct cgactaggtc tcggctgtac gcacctgagg 2220

gcccggcttc cgtggggagc cgggccctca gcatgtgcgc gccgtccacc gagcatgtgg 2280

aggccgtcgg ctgctgcgta tgcgcgacgt gatgagctgg atcactctgg gtattccatg 2340

accgaattcg ag 2352

Claims (10)

1. an albumen is following 1) or 2) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

2) with the amino acid residue sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with Avid kyowamycin produce Avrmectin relevant by 1) deutero-protein.

2. the described proteic encoding gene of claim 1.

3. gene as claimed in claim 2 is characterized in that: described gene is following 1) or 2) or 3):

1) encoding sequence is shown in sequence in the sequence table 3;

2) under stringent condition with 1) gene recombination and the described proteic gene of coding claim 1;

3) with 1) gene have the homology 90% or more and the described proteic gene of claim 1 of encoding.

4. the recombinant vectors, expression cassette or the transgenic cell line that contain claim 2 or 3 described genes.

5. recombinant vectors as claimed in claim 4 is characterized in that: described recombinant vectors is that the dna fragmentation that will contain promotor and claim 2 or 3 described genes inserts in the multiple clone site of plasmid pSET152 the recombinant expression vector that obtains; The nucleotide sequence of the described dna fragmentation that contains promotor and claim 2 or 3 described genes is shown in sequence in the sequence table 5.

6. the reorganization bacterium that contains claim 2 or 3 described genes.

7. reorganization bacterium as claimed in claim 6 is characterized in that: described reorganization bacterium is that claim 4 or 5 described recombinant vectorss are imported the reorganization bacterium that obtains in the purpose Avid kyowamycin.

8. reorganization bacterium as claimed in claim 7 is characterized in that: described purpose Avid kyowamycin is Avid kyowamycin (Streptomyces avermitilis) ZLX6003 CGMCC № .3229.

9. as arbitrary described reorganization bacterium among the claim 6-8, it is characterized in that: described reorganization bacterium is Avid kyowamycin (Streptomyces avermitilis) ZLX6056 CGMCC № .3796.

10. the application of arbitrary described reorganization bacterium in producing Avrmectin among the described albumen of claim 1, claim 2 or 3 described genes or the claim 6-9.

Images