CN102241750B - 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

Avrmectin (avermectin, AVM) is a kind of macrolide antibiotics being produced by Avid kyowamycin (Streptomyces avermitilis) fermentation, 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 to good killing action, and insecticidal activity exceeds tens times than general chemical pesticide.The mechanism of action of Avrmectin is different from conventional chemical sterilant, and its action target spot is the parasitic nerve conduction medium γ-aminobutyric acid 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 is decomposed into non-toxic substance by microorganism very soon in soil.Due to these outstanding advantages of Avrmectin, it is acknowledged as a kind of the most microbe-derived sterilant, therefore it agricultural, forestry, medicine and for animals on there is boundless market outlook and using value.Simultaneously take Avrmectin as parent, can also develop that a series of activity are higher, selectivity is stronger, use safer derivative new variety, commercial kind comprises ivermectin, emaricin, doractin, Ai Polinuo rhzomorph and Sai La rhzomorph etc.In addition, there is again in recent years investigator to find that avermitilis strain have antineoplastic effect, and the resistance of tumour cell is also had to 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, how the problems such as production cost height, improve the output of Avrmectin, reduces production costs, and the Agriculture Production for China is played to promoter action.In addition, be not only Avrmectin, how screening and optimizing bacterial strain, productive rate and raw material availability are realized maximize is the common problem that China's microbial fermentation engineering faces.At present mainly comprise two large classes for the method for the microbiotic output that improves streptomycete, a class is traditional method for mutation breeding, by physico-chemical process, bacterial strain is carried out to mutagenesis, then in mutagenic progeny, screens superior strain; Another kind of is by genetic method, and bacterial strain is carried out to direct hereditary change, improves antibiotic output.Obtain certain achievement although bacterial strain is carried out to mutagenesis by physico-chemical process, obtain 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, screening operation more complicated, there is certain blindness, in introducing favorable variation, may also can produce a lot of harmful variations, and the variation of harmful bacterial classification tends to affect optimization and the amplification of fermenting process; These traditional strain improvement modes are owing to understanding the Basic of Biology that causes changes of function, therefore more difficult other bacterial classifications that is applied to.Along with deepening continuously of bacterial genomes research, constantly deep to the understanding of bacterial gene function, people more and more tend to by genetics means, bacterial strain be transformed.Gene known function can be transformed by genetic method, strengthen the specific aim of operation, and workload is reduced greatly, screening time is also shortened.Therefore, transform Avid kyowamycin to improve the output of Avrmectin by genetic engineering means, have great importance.

Calendar year 2001 Kitasato institute has completed the gene order-checking of Avid kyowamycin, carries out sequence and functional analysis to being wherein responsible for Avrmectin biological synthesis gene cluster, and this gene cluster total length 82kb, has 18 open reading frames.There are 4 large 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, relevant with the modification of polyketide; In the upstream of the contiguous aveA4 in the right side of gene cluster, be a set of 8 genes (aveB I-aveBV III) that relate to the synthetic of olea disaccharides and shift; The upstream (left) of next-door neighbour aveA1 is the aveD of coding C5-O-methyltransgerase, and the OH of the responsible C5 that methyl is given to Avrmectin B above forms Avrmectin A.The downstream of aveF next-door neighbour aveD, the two transcriptional orientation is consistent, 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.

In microbiotic production process, the molecular regulation of gene level plays vital effect, by genetic method, streptomycete is transformed, and improves microbiotic output.Mainly that the regulatory gene producing by transformation microbiotic is carried out, have not only relevant to microbiotic biosynthesizing but also with Morphological Differentiation relevant regulatory gene, as bldA, relC, relA etc.; Also there is the biosynthetic regulatory gene of overall importance of the microbiotic of participation, as absA, absB, afsR etc.; Participate in addition the biosynthetic specificity regulatory gene of microbiotic, as ActII-ORF4, aveR, dnrI, redD, sanG, ccaR etc.To microbiotic, biosynthesizing plays the gene of positive control, can improve its expression amount and improve antibiotic output by improving its copy number in bacterial strain or changing promotor, to playing the gene of down regulation, can improve antibiotic output by knocking out this gene.But, the often less stable of bacterial strain getting by improving the method for copy number, because the Genetic carrier of high copy is often unstable, easily lose, if consider the industrial production in later stage, may more trials be incorporated on karyomit(e) the more stable stable existence of high yield genes involved at engineering strain that maintain by homologous recombination.Transform to can regulate and control the regulatory gene of Multiple Classes of Antibiotics output simultaneously that only to regulate and control the regulatory gene of single microbiotic output than transformation more effective.

In Avid kyowamycin, the mutant strain improving obtaining doractin " 1 " component concentration after aveC random mutation; To approach specificity regulatory gene aveR, increase copy number and do not improve Avrmectin output, make on the contrary host strain no longer produce Avrmectin.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, researchist is also devoted to find other regulatory gene, particularly regulatory gene 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 albumen relevant to Avid kyowamycin production Avrmectin.

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

1) protein being formed by the aminoacid sequence shown in sequence in sequence table 4;

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

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

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) albumen in can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned 2) encoding gene of the albumen in can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 3, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

Within the encoding gene (the hrdB gene after sudden change) of above-mentioned albumen also belongs to protection scope of the present invention.

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

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

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

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

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

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

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

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

Within the expression cassette that contains above-mentioned gene or transgenic cell line also belong to protection scope of the present invention.

Within the recombinant bacterium that contains above-mentioned gene also belongs to protection scope of the present invention.

Further, above-mentioned recombinant bacterium is that above-mentioned recombinant vectors is imported to the recombinant bacterium obtaining in object Avid kyowamycin.

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's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute) on 08 18th, 2009.ZLX6003 is the Avrmectin superior strain obtaining by traditional breeding way seed selection, has been applied to production industrial, produces grey spore on flat board.

Further, above-mentioned recombinant 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 and understands common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), and preserving number is CGMCC No.3796.There are 6 mutating acids in the sudden change HrdB in engineering strain ZLX6056, is respectively A137S, K139E, E163G, M356V, V357A, M389I.

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

Within the application in production Avrmectin of above-mentioned albumen, above-mentioned gene and arbitrary above-mentioned recombinant bacterium also belongs to protection scope of the present invention.

The present invention's RNA polymerase Sigma Factors in confirmation Avid kyowamycin increases production and has on direct acting basis Avrmectin, by its encoding gene hrdB orthogenesis is built to mutational vector storehouse, and import in Avid kyowamycin screening and obtain the recombinant bacterial strain that output improves, wherein synergy gene hrdB is for being applicable to the biosynthetic Optimal State of Avrmectin.

Specifically adopt and build with the following method engineering bacteria, design primer is by the hrdB gene with self promotor in pcr amplification Avrmectin wild mushroom, build the integrated expression plasmid containing hrdB gene, and the recombinant plasmid transformed building is obtained to recombinant bacterium storehouse in Avid kyowamycin bacterial strain, obtain by the method for high flux screening the recombinant bacterium that output improves.The above expression vector is integrated expression vector, and 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 mutation library is transformed to Avid kyowamycin is preferably the protoplast transformation method that PEG mediates, for improving transformation efficiency, the method that can directly transform by electricity in the time building is transformed into mutation expression carrier storehouse in the intestinal bacteria ET12567 of restriction modification effect defect, collects resistance bacterium colony and therefrom extract plasmid the protoplastis that transforms again Avid kyowamycin; But also can adopt method conventional in other bioengineering field, such as electrotransformation, conjugal transfer method etc.

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

Engineering bacteria ZLX6056 of the present invention, growth curve on 180 tons of tanks 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.

The σ relating to for the present invention in work of the present invention ^hrdBfor the main Sigma Factors in streptomycete, be mainly responsible for the regulation and control of primary metabolite gene transcription level.The complete sequence of encoding gene hrdB gene and both sides fragment sequence can be retrieved and obtain in public database, be positioned at the base of from 2976855 to 2978393 (complementary strands) on genome, its coded protein sequence is numbered NP_823620 in Genbank database, is for No. GI 29606091.Although Avid kyowamycin genome sequence is concurrent table after measured, the complete sequence of hrdB can obtain in Genbank database, but, about this gene for the biosynthetic impact of secondary metabolism of Streptomyces product unclear, in existing document, also do not utilize this gene to improve the report of yield of streptomycete antibiotic.The present invention has confirmed that hrdB is for the biosynthetic regulating and controlling effect of Avrmectin by experiment, and utilizes the genetic manipulation of this gene to improve the output of Avrmectin.

Accompanying drawing explanation

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

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

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

Fig. 4 is that suddenly change in the Avid kyowamycin sudden change Producing Strain ZLX6056 mutational site of Sigma Factors is analyzed.

Fig. 5 is the fermentation unit graphic representation of industrial strain ZLX6003 and sudden change Producing Strain ZLX6056 of setting out.

Embodiment

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

In 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

Design primer, is positioned at the hrdB structural gene sequence on genomic dna, upstream primer HBNde01:5 '-GC for increasing 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, base with underscore is restriction enzyme NdeI and SalI recognition site, and amplified production should be 1540bp.

Take the genomic dna of Avid kyowamycin ZLX6003 (being preserved in CGMCC, NO.3229) as template, take HBNde01 and HBSal02 as primer, carry out pcr amplification, adopt 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 circulations; 72 ℃, 10min.Amplified production is carried out to agarose gel electrophoresis detection, have a specific amplified band at about 1.5kb place.

2, HrdB protein expression vector builds

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 order-checking comparison, the fragment increasing is hrdB gene structure gene (encoding sequence as shown in sequence in sequence table 1, the albumen shown in sequence 2 in codified sequence table) really.By the T carrier that contains hrdB structure gene after NdeI and SalI enzyme are cut, reclaim the hrdB fragment of 1.5kb, this fragment is connected with the carrier pET28b (Novagen) cutting with XhoI enzyme through NdeI, connects the competent cell of product conversion escherichia coli DH5a.From transformant, extract plasmid and carry out enzyme and cut checking, by correct recombinant plasmid called after pZY165 respectively.

Two, protein expression and purification and the HrdB albumen impact of transcribing on aveR

1, HrdB protein expression and purifying

Choose mono-clonal (containing corresponding microbiotic) in the LB liquid nutrient medium of 5ml from flat board, 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.Under non-Denaturing after ultrasonication cell, carry out after preliminary purification with the HisTrap HP affinity column (GE Healthcare) of 5ml, after suction filtration is concentrated, carry out molecular sieving.Result as shown in Figure 1A for albumen after purifying (HrdB albumen).

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

The quantitative HrdB albumen of JiangRNA center enzyme and purifying mixes by 1: 1 concentration ratio, and polysaccharase mixture is placed on 30 ℃ of water-bath 5min; Add 3.5ul to contain [a-32P] CTP (400Ci/mmol) substrate mixture and continue temperature bath 15 minutes; After reflection finishes, add sample-loading buffer to carry out 5%polyacrylamide gel (containing 7M urea) electrophoresis detection, after radioautograph, result as shown in Figure 1B.

Fig. 2 result shows HrdB albumen specific identification aveR upstream promoter sequence in vitro, and start HrdB albumen transcribing aveR, and along with the increase of HrdB protein concentration, the amount of the transcription product of aveR presents and increases progressively trend, and 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

The structure of the recombinant plasmid that, contains hrdB gene and himself promotor

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

Design primer, is positioned on Avid kyowamycin karyomit(e) the hrdB gene [NC_003155.4, complement (2976855..2978393)] containing self promotor, upstream primer PodP1:5 '-CAC for pcr amplification 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 with underscore is restriction enzyme XbaI and EcoRI recognition site, and amplified production should be 2352bp.

Take total DNA of Avid kyowamycin wild type strain ATCC31267 as template, take PodP1 and PodP2 as primer, adopt LA-Taq enzyme and the GC buffer I of TaKaRa company to carry out pcr amplification, amplification condition is 95 ℃, 3min; (95 ℃, 1min; 64.6 ℃, 1min; 72 ℃, 2.5min) × 25 circulations; 72 ℃, 10min.Amplified production is carried out to agarose gel electrophoresis detection, have a specific amplified band 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 order-checking comparison, the fragment increasing is the hrdB gene containing self promotor really.By the T carrier that contains hrdB gene after XbaI and EcoRI enzyme are cut, reclaim the hrdB fragment of 2.3kb, carrier pSET152 (the Bierman M cutting by this fragment with 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) be connected, connect the competent cell of product conversion bacillus coli DH 5 alpha.From transformant, extract plasmid and carry out enzyme and cut checking, by correct recombinant plasmid called after pZY126 respectively.The plasmid map of pZY126 as shown in Figure 2.

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

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

Design primer, be positioned at the hrdB structural gene sequence on pZY126 plasmid for increasing, 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.

Take the pZY126 plasmid DNA after purifying as template, take PodP3 and PodP4 as primer, carry out pcr amplification, adopt the Mutazyme II DNA Polymerase enzyme of Stratagene company to carry out pcr amplification, amplification condition is 95 ℃, 2min; (95 ℃, 1min; 59 ℃, 1min; 72 ℃, 1.5min) × 30 circulations; 72 ℃, 10min.Amplified production is carried out to agarose gel electrophoresis detection, have a specific amplified band at about 1.6kb place.

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

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

Take PCR product after above-mentioned purifying as primer, according to preparing reaction system in GeneMorph II EZClone product description, amplification condition is 95 ℃, 1min; (95 ℃, 50sec; 60 ℃, 50sec; 68 ℃, 3min) × 25 circulations; After finishing, reaction places on ice 2 minutes.

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

So far the pZY126* expression plasmid carrier storehouse of, containing sudden change hrdB fragment has built.

The conversion in embodiment 3, mutant plasmid storehouse

One, the amplification in mutant plasmid storehouse

Owing to there being very strong restriction modification effect in Avid kyowamycin, directly transform Avid kyowamycin with extracting from the plasmid of E.coli DH5 α, transformation efficiency is extremely low, sometimes even can not get transformant.And use from the plasmid of recipient bacterium E.coli ET12567 that there is no restriction modification effect, its transformation efficiency obviously improves.Therefore, the mutant plasmid storehouse building and control plasmid are first transformed into respectively to E.coli ET12567 (pUZ8002), and (non-patent literature of recording 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) to obtain non-methylated DNA, and then transform the protoplastis of Avid kyowamycin by non-methylated plasmid DNA.

The competent cell that contains suddenly change the pZY126* expression plasmid carrier storehouse of hrdB fragment and original plasmid pZY126 in contrast and pSET152 electric shock Transformed E .coli ET12567 will be built 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 flat board visible bacterium colony, collect after all bacterium colonies with fresh liquid LB, add 15% glycerine to be saved in-80 ℃, or directly access contains in corresponding antibiotic LB and cultivates further amplification plasmid, extracts plasmid DNA, enzyme is cut checking.

Two, mutant plasmid storehouse transforms Avid kyowamycin

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

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

The preparation of the preparation of colibacillary conversion in the present embodiment, Avid kyowamycin protoplastis and method for transformation, RM 14 and MS substratum referring to the Ph D dissertation of Zhang Xiaolin (Zhang Xiaolin. the genetic modification of polyketide synthase gene in Avid kyowamycin. 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 transformants thereof grow after abundant spore on slant medium, Avrmectin yield level to 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, its access being equipped with to the seed bottle of the sterilized seed culture medium of 40mL, 28 ℃ of shaking tables are 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) in the triangular flask that the sterilized fermention medium of 30mL is housed, 28 ℃ of shaking tables are cultivated 10 days, put bottle, measure the fermentation unit of Avrmectin by HPLC method, concrete HPLC is analyzed as follows described in the step 2 of stating.

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 of preparing Avrmectin).

2, the HPLC of tunning analyzes

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

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

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

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

The fermentation results of Fig. 3 shows, the output of the Avrmectin output after the conversion starting strain ZLX6003 of control plasmid pZY126 (representing with pSET152-hrdB in 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, 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 common micro-organisms center, preserving number is CGMCCNo.3796.

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

1, 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 after ethanol deposition and purification endonuclease bamhi from connecting, will certainly connect product and transform competent escherichia coli cell, be applied to the LB flat board that contains apramycin resistance, 37 ℃ are cultured to and occur white colony.Picking list bacterium colony extracts plasmid DNA after cultivating, after XbaI and EcoRI enzyme are cut, reclaim 2.3kb containing promotor in interior sudden change hrdB gene fragment, after being connected with the carrier pUC18 cutting through same enzyme, the gene fragment of this 2.3kb transforms bacillus coli DH 5 alpha competent cell, be applied to the LB flat board that contains amicillin resistance, 37 ℃ are cultured to and occur white colony.Picking list bacterium colony extracts plasmid DNA after cultivating, and send Beijing Hua Da company to check order after enzyme is cut checking.Sequencing result shows, the nucleotide sequence of the gene fragment of this 2.3kb is as shown in sequence in sequence table 5.Wherein the nucleotide sequence from shown in the 658-2197 position of 5 ' end of sequence 5 is consistent with sequence in sequence table 3, is the sequence of the hrdB gene after sudden change, the hrdB albumen in codified sequence table after the sudden change shown in sequence 4.

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

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

1) structure of recombinant expression vector

By the 2.3kb reclaiming in step 1 containing promotor, at interior sudden change hrdB gene fragment and the carrier pSET152 after XbaI, EcoRI double digestion, (non-patent literature of recording this material is Bierman M, 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.Then (non-patent literature of recording this material is MacNeil DJ this recombinant expression vector to be transformed to intestinal bacteria E.coli ET12567, 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 occur white colony.Picking list bacterium colony extracts plasmid DNA after cultivating, and cuts the laggard pacing order of checking through enzyme, shows that the recombinant expression vector obtaining is that in sequence table, the DNA fragmentation described in sequence 5 is inserted between the XbaI of pSET152 and EcoRI restriction enzyme site and forms.

2) transform Avid kyowamycin ZLX6003 bacterial strain and ferment

By step 1) recombinant expression vector that obtains is transformed into the protoplastis of Avid kyowamycin ZLX6003 bacterial strain, be applied on RM 14 flat boards of the not added with antibiotic having dried up, cultivate after 16-20h for 28 ℃, on flat board, cover the aqueous solution of 1mL containing 1mg apramycin, continue to cultivate 7-10 days at 28 ℃, the bacterium colony growing is transformant.The random uniform transformant of picking colony form, is inoculated on the MS flat board containing 20ug/mL apramycin, cultivates for 28 ℃ and within 7 days, recovers to produce robe.。PCR verifies correct transformant called after pZY148-hrdB ^zLX6056, the transformant that then 5 PCR of random choose verified, in embodiment 4, the method for step 1 step 2 is fermented.

The protoplastis that simultaneously carrier pSET152 is transformed into Avid kyowamycin ZLX6003 bacterial strain, obtains empty carrier control strain.

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

Fermenting experiment repeats 3 times, result as shown in table 2 (the B1a output with starting strain ZLX6003 fermentation after 240 hours is decided to be 100%), recombinant bacterium pZY148-hrdB ^zLX6056ferment and can reach 148.23% after 240 hours, can recover growth and the Yield Characters 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 in sequence table, the hrdB gene after the sudden change shown in sequence 3 can improve Avrmectin output.

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

Embodiment 5, engineering strain genetic stability detect

Engineering strain ZLX6056 of the present invention is carried out to inclined-plane to go down to posterity, passed altogether for 5 generations, 28 ℃ are cultured to and grow (approximately 12～15 days) after vigorous spore, the bacterium that every culture obtains is all fermented according to the shake flask fermentation method in embodiment 4 step 1 steps 1, ferment and extract by method described in embodiment 4 step 1 steps 2, and calculating the ability of bacterial strain production Avermectin B1a.3 repetitions are established in experiment, and result is taken the mean.Result shows that engineering strain ZLX6056 of the present invention is 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 the engineering strain ZLX6056 that the present embodiment has compared starting strain ZLX6003 and contained the hrdB gene after sudden change on 180 tons of fermentor tanks 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 filling pocket, by formulated fermention medium and adjust pH, the substratum preparing is pumped into 180m ³fermentor tank in, after polyphony pH, 121 ℃～130 ℃ sterilizings 30 minutes～1 hour, are cooled to 28 degree after sterilizing, for subsequent use after polyphony pH.To be cultured to the seed of logarithmic phase, under aseptic condition by 10m ³(± 20%) left and right seed liquor pumps in above-mentioned sterilized fermentor tank, start fermentation culture program, 28～29 ℃ of cultivations, control DO in 50% left and right, pH is controlled at 7.8 left and right, stirring velocity is 0～100rpm in earlier stage, and 24h is 100～200rpm later, after 72 hours, is about 200～300rpm.Regularly detect mycelial growth and produce plain situation, fermentor tank is put tank after moving approximately 312 hours.

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 of preparing Avrmectin).

Result as shown in Figure 5, engineering strain ZLX6056 still can keep the advantage of increase of production on large scale fermentation tank after 200 hours, in 312 hours, the ability of engineering strain production Avermectin B1a of the present invention reaches 6382 μ g/ml fermented liquids, improves 53.1% compared with the product element ability of starting strain ZLX6003 bacterial strain (4167 μ g/ml).

Sequence table

<110> Institute of Microorganism, Academia Sinica

Mono-kind of <120> produces gene engineering method and the special strain therefore thereof of 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 (12)

1. an albumen, the protein being formed by the aminoacid sequence shown in sequence in sequence table 4.

2. the encoding gene of albumen claimed in claim 1.

3. encoding gene as claimed in claim 2, is characterized in that: the nucleotide sequence of described encoding gene is as shown in sequence in sequence table 3.

4. contain the recombinant vectors of encoding gene described in claim 2 or 3.

5. recombinant vectors as claimed in claim 4, is characterized in that: described recombinant vectors is that the DNA fragmentation that contains encoding gene described in promotor and claim 2 or 3 is inserted in the multiple clone site of plasmid pSET152, the recombinant expression vector obtaining; The described nucleotide sequence that contains the DNA fragmentation of encoding gene described in promotor and claim 2 or 3 is as shown in sequence in sequence table 5.

6. contain the expression cassette of encoding gene described in claim 2 or 3.

7. contain the transgenic cell line of encoding gene described in claim 2 or 3.

8. contain the recombinant bacterium of the encoding gene described in claim 2 or 3.

9. recombinant bacterium as claimed in claim 8, is characterized in that: described recombinant bacterium is that the recombinant vectors described in claim 4 or 5 is imported to the recombinant bacterium obtaining in object Avid kyowamycin.

10. recombinant bacterium as claimed in claim 9, is characterized in that: described object Avid kyowamycin be Avid kyowamycin ( streptomyces avermitilis) ZLX6003, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC № .3229.

11. recombinant bacteriums as claimed in claim 8 or 9, is characterized in that: described recombinant bacterium be Avid kyowamycin ( streptomyces avermitilis) ZLX6056, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC № .3796.

Described in 12. claims 1 described in albumen, claim 2 or 3 in encoding gene or claim 8-11 arbitrary described recombinant bacterium in the application of producing in Avrmectin.

Images