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 abamectin and a special bacterial strain thereof. The protein provided by the present invention is a mutated HrdB protein, specifically the protein of the following 1) or 2): 1) a protein consisting of the amino acid sequence shown in sequence 4 in the sequence listing; 2) combining sequence 4 in the sequence listing A protein derived from 1) that has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to the production of abamectin by Streptomyces avermitilis. The recombinant bacterium obtained after the mutated hrdB gene is introduced into the ZLX6003 bacterial strain provided by the present invention can produce 5732.05±91.26 μg/ml of abamectin after 240 hours of shaking flask culture. The growth curve of the engineered bacterium ZLX6056 of the present invention on a 180-ton tank shows that the genetically engineered bacterium maintains the excellent shape of the original high-yield strain, and the yield reaches 6382ug/ml.

Description

A kind of genetic engineering method for producing abamectin and special bacterial strain thereof

technical field

The invention relates to a genetic engineering method for producing abamectin and a special bacterial strain thereof.

Background technique

Avermectin (avermectin, AVM) is a macrolide antibiotic produced by the fermentation of Streptomyces avermitilis, which has eight components (A1a, A1b, A2a, A2b, B1a, B1b, B2a, B2b), in which the B1a component has the strongest insecticidal activity, and has a good killing effect on nematodes, mites and arthropods, and its insecticidal activity is dozens of times higher than that of general chemical pesticides. The mechanism of action of abamectin is different from that of conventional chemical insecticides. Its target is the neurotransmitter γ-aminobutyric acid (GABA) of nematodes and arthropod parasites. It has very little toxicity to mammals. Sex is extremely high. The antibiotic has a very short residual time in plants and is quickly decomposed into non-toxic substances by microorganisms in the soil. Due to these outstanding advantages of Abamectin, it is recognized as the safest and most effective microbial-derived pesticide, so it has very broad market prospects and application value in agriculture, forestry, medicine and veterinary . At the same time, with abamectin as the parent, a series of new derivatives with higher activity, stronger selectivity and safer use can also be developed. The commercialized varieties include ivermectin, emamectin, dora Selamectin, epelinocetin, and selamectin. In addition, in recent years, some researchers have found that avermectin has anti-tumor effects, and it also has a certain inhibitory effect on the drug resistance of tumor cells.

At present, Abamectin has been industrialized in China and has achieved huge economic and social benefits. However, the production strains of abamectin in my country still have problems such as low fermentation unit and high production cost. How to increase the output of abamectin and reduce production cost will play a role in promoting the development of agricultural production in our country. In addition, not only abamectin, how to screen and optimize strains to maximize yield and raw material utilization is a common problem faced by microbial fermentation projects in my country. At present, the methods for improving the antibiotic production of Streptomyces mainly include two categories, one is the traditional mutation breeding method, the strain is mutated by physical and chemical methods, and then the high-yield strain is screened in the mutagenized progeny; the other is Through the method of genetics, direct genetic changes are made to the strains to increase the production of antibiotics. Although the mutagenesis of bacterial strains by physical and chemical methods has achieved certain results, and a large number of high-yield strains have been obtained, their shortcomings are also obvious, mainly including consuming a lot of manpower, material resources and time, and the screening work is more complicated. Certain blindness, while introducing beneficial mutations, may also produce many harmful mutations, and harmful strain variations often affect the optimization and amplification of the fermentation process; these traditional strain breeding methods are due to the biological The scientific basis is not understood, so it is difficult to popularize and apply to other strains. With the continuous deepening of bacterial genome research and the deepening understanding of bacterial gene functions, people are more and more inclined to use genetic means to modify bacterial strains. Genetic methods can be used to modify genes with known functions, which enhances the pertinence of operations, greatly reduces the workload, and shortens the screening time. Therefore, it is of great significance to improve the production of abamectin by transforming Streptomyces avermitilis by means of genetic engineering.

In 2001, the Kitasato Institute completed the genome sequencing of Streptomyces avermitilis, and performed sequence and functional analysis on the gene cluster responsible for the biosynthesis of avermectin. The gene cluster is 82kb in length and has 18 open reading frames. There are 4 large reading frames (AveA1-AveA2 and AveA3-AveA4) inside the gene cluster, encoding multifunctional polyketide synthases; aveC and aveE genes are located between aveA1-aveA2 and aveA3-aveA4 genes, and It is related to the modification; on the right side of the gene cluster, adjacent to the upstream of aveA4, is a set of 8 genes (aveB I-aveBV III) involved in the synthesis and transfer of oleanobiose; immediately upstream of aveA1 (left) is the encoding aveD of C5-O-methyltransferase is responsible for transferring the methyl group to the OH of C5 of avermectin B to form avermectin A. aveF is immediately downstream of aveD, and the transcription direction of the two is the same, which may belong to the same transcription unit. aveF encodes a C5 ketoreductase that catalyzes the production of avermectin B. aveR is located downstream of aveF (but the transcription direction is opposite), which belongs to the pathway-specific regulation gene, and is a positive regulation gene of the whole gene cluster of abamectin biosynthesis.

Molecular regulation at the gene level plays a vital role in the process of antibiotic production, and Streptomyces is modified by genetic methods to increase antibiotic production. It is mainly carried out by modifying the regulatory genes of antibiotic production. There are regulatory genes related to antibiotic biosynthesis and morphological differentiation, such as bldA, relC, relA, etc.; there are also global regulatory genes involved in antibiotic biosynthesis, such as absA , absB, afsR, etc.; there are also specific regulatory genes involved in antibiotic biosynthesis, such as ActII-ORF4, aveR, dnrI, redD, sanG, ccaR, etc. For genes that positively regulate the biosynthesis of antibiotics, the production of antibiotics can be increased by increasing its copy number in the strain or changing the promoter to increase its expression; for genes that play a negative regulatory role, the gene can be knocked out to increase the production of antibiotics. However, the strains obtained by increasing the copy number are often less stable, because the high-copy genetic carrier is often unstable and easy to lose. If considering the later industrial production, more attempts may be made to integrate into the chromosome through homologous recombination On the other hand, it is more stable to maintain the stable existence of high-yielding related genes in engineering strains. It is more effective to modify the regulatory gene that can simultaneously regulate the production of multiple antibiotics than to modify the regulatory gene that only regulates the production of a single antibiotic.

In Streptomyces avermitilis, a mutant strain with increased doramectin "1" component content can be obtained after random mutation of aveC; for the pathway-specific regulation gene aveR, increasing the copy number did not increase the production of avermectin, but instead Make the host strain no longer produce abamectin. There are negative regulatory genes aveR1 and aveR2 upstream of the aveR gene, and the inactivation of any one or both of these two genes will greatly increase the production of abamectin. (US Patent US6197591, Stutzman-Engwall). In addition to the regulatory genes located in the abamectin biosynthetic gene cluster, researchers are also committed to finding other regulatory genes, especially global regulatory genes (global regulatory genes), such as afsR2 and orfX, so as to more effectively improve the abamectin output.

Contents of the invention

The object of the present invention is to provide a protein related to the production of abamectin by Streptomyces avermitilis.

The protein provided by the present invention is a mutated HrdB protein, specifically the following 1) or 2) protein:

1) A protein consisting of the amino acid sequence shown in Sequence 4 in the sequence listing;

2) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid residue sequence of Sequence 4 in the sequence listing and the protein derived from 1) related to the production of abamectin by Streptomyces avermitilis .

In order to make the protein in 1) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 4 in the sequence listing can be linked with the tags shown in Table 1.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in the above 2) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The gene encoding the protein in the above 2) can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 3 in the sequence listing, and/or carrying out one or several base pairs of missense mutations , and/or connect the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The above protein coding gene (mutated hrdB gene) also falls within the protection scope of the present invention.

The above-mentioned gene can be the following 1) or 2) or 3):

1) The coding sequence is shown in sequence 3 in the sequence listing;

2) a gene that hybridizes with the gene of 1) under stringent conditions and encodes the protein;

3) A gene that has more than 90% homology with the gene in 1) and encodes the protein.

Sequence 3 is the sequence of the mutated hrdB gene (1539 nucleotides), which can encode the mutated hrdB protein shown in Sequence 4 in the sequence list.

The above-mentioned stringent conditions can be 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65° C. in DNA or RNA hybridization experiments and membrane washing.

Recombinant vectors containing the above genes also fall within the protection scope of the present invention.

Further, the above-mentioned recombinant vector can specifically be a recombinant expression vector obtained by inserting the DNA fragment containing the promoter and the above-mentioned gene into the multiple cloning site of the plasmid pSET152; the nucleoside of the DNA fragment containing the promoter and the above-mentioned gene The acid sequence is shown as sequence 5 in the sequence listing.

Expression cassettes or transgenic cell lines containing the above genes also fall within the protection scope of the present invention.

Recombinant bacteria containing the above-mentioned genes also fall within the protection scope of the present invention.

Further, the above-mentioned recombinant bacteria are recombinant bacteria obtained by introducing the above-mentioned recombinant vector into the target Streptomyces avermitilis.

The above-mentioned Streptomyces avermitilis can be Streptomyces avermitilis ZLX6003CGMCC №.3229. ZLX6003 was deposited on August 18, 2009 in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China). ZLX6003 is a high-yielding avermectin strain obtained through traditional breeding methods, which has been applied in industry and produces gray spores on plates.

Further, the above-mentioned recombinant bacteria is Streptomyces avermitilis ZLX6056 CGMCC №.3796. ZLX6056 was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on May 4, 2010, and the preservation number is CGMCC No. 3796. The mutant HrdB in the genetically engineered strain ZLX6056 has 6 mutant amino acids, which are A137S, K139E, E163G, M356V, V357A, and M389I.

Both ZLX6003 and ZLX6056 are aerobic Gram-positive mesophilic actinomycetes that form branched basal hyphae and long, tightly spiraled aerial hyphae. The spore chain consists of 15 or more round or oval spores with a smooth surface. Gray aerial spore clusters were formed on the oat medium, and the back of the colony was dark brown. Production of melanin on protein-aged yeast extract and iron media.

The application of the above-mentioned protein, the above-mentioned gene and any one of the above-mentioned recombinant bacteria in the production of abamectin also falls within the protection scope of the present invention.

In the present invention, on the basis of confirming that the RNA polymerase σ factor in Streptomyces avermitilis has a direct effect on increasing the production of abamectin, a mutation carrier library is constructed by directed evolution of its coding gene hrdB, and introduced into Streptomyces avermitilis for screening to obtain The recombinant strain with improved yield, wherein the synergistic gene hrdB is in an optimized state suitable for avermectin biosynthesis.

Specifically adopt the following method to construct engineering bacteria, design primers to amplify the hrdB gene with its own promoter in the abamectin wild bacterium by PCR, construct an integrated expression plasmid containing the hrdB gene, and transform the constructed recombinant plasmid into Abamectin The recombinant bacteria library was obtained from the Streptomyces veinensis strain, and the recombinant bacteria with improved yield were obtained through high-throughput screening. The above-mentioned expression vector is an integrated expression vector, which can be stably applied in production, and the starting vector is the Escherichia coli-Streptomyces shuttle vector pSET152.

The method of transforming the hrdB gene expression carrier mutant library into Streptomyces avermitilis is preferably PEG-mediated protoplast transformation. In order to improve the transformation efficiency, the mutant expression carrier library can be directly transformed into a restriction modification by the method of electroporation during construction. In the deficient Escherichia coli ET12567, the resistant colonies were collected and plasmids were extracted from them to transform the protoplasts of Streptomyces avermitilis; however, other commonly used methods in the field of bioengineering, such as electroporation and conjugative transfer, can also be used.

The starting bacterial strain that is used to construct recombinant bacterium can be the bacterial strain of any Streptomyces avermitilis, considers that high-yield bacterial strain is more difficult to improve output by traditional mutagenesis method, in the present invention with existing abamectin high-yield industrial strain (Avermectin Streptomyces avermitilis (ZLX6003 CGMCC 3229) was the starting strain. The recombinant bacterium obtained after the mutated hrdB gene provided by the present invention is introduced into the ZLX6003 bacterial strain can reach 5732.05 ± 91.26 μ g/ml of abamectin output after 240 hours of shaking flask culture, which is the peak of the abamectin output of the starting bacterial strain. 148.23%.

The growth curve of the engineering bacterium ZLX6056 of the present invention on a 180-ton tank shows that the genetically engineered bacterium maintains the excellent shape of the original high-yield strain, and the output reaches 6382ug/ml, which can be well adapted to large-scale fermentation and can be directly used for Avermella The fermentative production of abamectin increases the output of abamectin, thereby reducing production costs.

The σ ^hrdB involved in the present invention is the main σ factor in Streptomyces, which is mainly responsible for the regulation of primary metabolic gene transcription level. The full sequence of the coding gene hrdB gene and the sequence of the fragments on both sides can be retrieved in public databases, located at bases from 2976855 to 2978393 (on the complementary strand) on the genome, and the protein sequence encoded by it is numbered NP_823620 in the Genbank database , GI number 29606091. Although the genome sequence of Streptomyces avermitilis has been determined and published, and the full sequence of hrdB is available in the Genbank database, the effect of this gene on the biosynthesis of Streptomyces secondary metabolites is not clear, and in the existing literature However, there is no report on using this gene to increase the production of Streptomyces antibiotics. The invention confirms the regulating effect of hrdB on the biosynthesis of avermectin through experiments, and utilizes the genetic manipulation of the gene to increase the output of abamectin.

Description of drawings

Figure 1 HrdB protein in vitro expression and purification and in vitro transcription test to demonstrate its regulation of abamectin biosynthesis; 1A) HrdB protein in vitro induced expression and purification, 1B) in vitro transcription test to confirm its regulation of abamectin biosynthesis.

Fig. 2 Plasmid map of recombinant plasmid pZY126 containing hrdB gene and its own promoter.

Fig. 3 is the abamectin fermentation unit of high-yielding industrial Streptomyces avermitilis and its different transformants.

Fig. 4 is an analysis of the mutation site of the mutant σ factor in the mutant high-yielding strain of Streptomyces avermitilis ZLX6056.

Fig. 5 is the fermentation unit curve of the starting industrial strain ZLX6003 and the mutant high-yielding strain ZLX6056.

Detailed ways

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

In the following examples, unless otherwise specified, all are conventional methods.

Embodiment 1, the regulatory effect of HrdB protein on Abamectin biosynthesis-specific regulatory gene aveR transcription

1. Expression of HrdB protein in vitro

1. Amplification and purification of hrdB structural gene

Design primers for amplifying the hrdB structural gene sequence located on genomic DNA, the upstream primer HBNde01: 5′-GC CATATG TCGGCCAGCACATCC-3′, located at the start codon ATG position of the hrdB gene, and the downstream primer HBSal02: 5′--GCGTCGACTAGTCGAGGTAGTCGC -3', located at the stop codon TAG of the hrdB gene, the underlined bases are the recognition sites of restriction endonucleases NdeI and SalI, and the amplified product should be 1540bp.

Using the genomic DNA of Streptomyces avermitilis ZLX6003 (preserved in CGMCC, NO.3229) as a template, using HBNde01 and HBSal02 as primers, PCR amplification was carried out, and LA-Taq enzyme and GC buffer I from TaKaRa Company were used to perform PCR amplification. The amplification conditions were 95°C, 3min; (95°C, 1min; 60°C, 1min; 72°C, 1.5min) x 25 cycles; 72°C, 10min. The amplified product was detected by agarose gel electrophoresis, and there was a specific amplified band at about 1.5kb.

2. Construction of HrdB protein expression vector

Use the agarose gel recovery kit to cut the gel to recover the PCR amplification product, and connect it to the T carrier pMD 18-T (TaKaRa company). After sequencing and comparison, it shows that the amplified fragment is indeed the hrdB gene structural gene (coding sequence such as As shown in sequence 1 in the sequence listing, it can encode the protein shown in sequence 2 in the sequence listing). After the T vector containing the hrdB structural gene was digested with NdeI and SalI, the 1.5kb hrdB fragment was recovered, and this fragment was connected with the vector pET28b (Novagen) digested with NdeI and XhoI, and the ligation product was transformed into a competent Escherichia coli DH5a cell. Plasmids were extracted from transformants for enzyme digestion verification, and the correct recombinant plasmids were named pZY165.

2. Protein expression and purification and the effect of HrdB protein on the transcription of aveR

1. HrdB protein expression and purification

Pick a single clone from the plate and put it into 5ml LB liquid medium (containing corresponding antibiotics), culture overnight to the logarithmic growth phase; add IPTG to a final concentration of 0.4mM and continue to culture for 10h, take 200ul of the bacterial liquid for SDS-PAGE gel electrophoresis Detection of protein expression. After the cells were sonicated under non-denaturing conditions, they were initially purified with a 5ml HisTrap HP affinity column (GE Healthcare), concentrated by suction, and then separated by molecular sieves. The results of the purified protein (HrdB protein) are shown in Figure 1A.

2. The specific regulatory effect of HrdB protein on aveR

Mix the RNA central enzyme and the purified quantitative HrdB protein at a concentration ratio of 1:1, and place the polymerase mixture in a water bath at 30°C for 5 minutes; add 3.5ul of the substrate mixture containing [a-32P]CTP (400Ci/mmol) and continue to incubate for 15 minutes ; After the reaction, add loading buffer for 5% polyacrylamide gel (containing 7M urea) electrophoresis detection, and the results after autoradiography are shown in Figure 1B.

The results in Figure 2 show that HrdB protein can specifically recognize the upstream promoter sequence of aveR in vitro, and initiate the transcription of aveR by HrdB protein, and with the increase of HrdB protein concentration, the amount of aveR transcripts shows an increasing trend, the results show that HrdB The protein can participate in the regulation of the transcription level of aveR.

The in vitro transcription test method refers to the literature (Hahn MY, Bae JB, Park JH, Roe JH. Isolation and characterization of Streptomyces coelicolor RNA polymerase, its sigma, and antisigmafactors. Methods Enzymol 2003, 370: 73-82).

Embodiment 2, the construction of hrdB gene expression vector

1. Construction of recombinant plasmid containing hrdB gene and its own promoter

1. Amplification of the hrdB gene containing its own promoter

Design primers for PCR amplification of the hrdB gene [NC_003155.4, complement (2976855..2978393)] located on the chromosome of Streptomyces avermitilis with its own promoter, and the upstream primer PodP1: 5′-CAC TCTAGA CCCTGAGGTGGAGCGTGTG-3′ is located in 649bp upstream of the hrdB start codon, the downstream primer PodP2: 5′-CTC GAATTC GGTCATGGAATACCCAGAGTGAT-3′, located 120bp downstream of the hrdB stop codon, the underlined bases are the recognition sites of restriction enzymes XbaI and EcoRI, The amplification product should be 2352bp.

Using the total DNA of Streptomyces avermitilis wild-type strain ATCC31267 as a template, using PodP1 and PodP2 as primers, the LA-Taq enzyme and GC buffer I of TaKaRa Company were used for PCR amplification. The amplification conditions were 95°C, 3min; (95 ℃, 1min; 64.6℃, 1min; 72℃, 2.5min)×25 cycles; 72℃, 10min. The amplified product was detected by agarose gel electrophoresis, and there was a specific amplified band at about 2.3 kb.

2. Construction of recombinant plasmid pZY126

The agarose gel recovery kit was used to cut the gel to recover the PCR amplified product, and connect it to the T vector pMD 18-T (TaKaRa company). The sequencing comparison showed that the amplified fragment was indeed the hrdB gene containing its own promoter. After the T vector containing the hrdB gene was digested with XbaI and EcoRI, the 2.3 kb hrdB fragment was recovered, and this fragment was digested with the vector pSET152 (Bierman M, Logan R, O'Brien K, et al. Plasmid cloning vectors For the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43-49), the ligation product was transformed into competent cells of Escherichia coli DH5α. Plasmids were extracted from transformants for enzyme digestion verification, and the correct recombinant plasmids were named pZY126. The plasmid map of pZY126 is shown in FIG. 2 .

2. Construction of hrdB gene mutation library based on pZY126 plasmid

1. Amplification and purification of mutant hrdB structural gene

Design primers for amplifying the hrdB structural gene sequence located on the pZY126 plasmid, the upstream primer PodP3: 5'-TGTTCGTGTCGGCCAGCAC-3', located 5bp upstream of the start codon of the hrdB gene, and the downstream primer PodP4: 5'-TGCGTACAGCCGAGACCTAGTC-3 ', located 14 bp downstream of the stop codon of the hrdB gene, the amplified product should be 1560 bp.

Using the purified pZY126 plasmid DNA as a template, using PodP3 and PodP4 as primers, PCR amplification was carried out, and Stratagene’s Mutazyme II DNA Polymerase enzyme was used for PCR amplification. The amplification conditions were 95°C, 2min; (95°C, 1min ; 59°C, 1min; 72°C, 1.5min) × 30 cycles; 72°C, 10min. The amplified product was detected by agarose gel electrophoresis, and there was a specific amplified band at about 1.6 kb.

The PCR amplification product was purified and recovered using a PCR product purification kit, and 1 ul of the purified product was subjected to agarose gel electrophoresis and nanodrop detection for quantification of the purified product.

2. Construction of the pZY126* plasmid fragment library containing the mutated hrdB fragment

Using the above-mentioned purified PCR products as primers, prepare a reaction system according to the GeneMorph II EZClone product manual, and the amplification conditions are 95°C, 1min; (95°C, 50sec; 60°C, 50sec; 68°C, 3min) × 25 cycles; Place on ice for 2 minutes after the reaction.

DpnI enzyme is added to the amplified product to digest the plasmid DNA originally used as a template in the amplified product.

So far, the pZY126* expression plasmid vector library containing the mutant hrdB fragment has been constructed.

Embodiment 3, transformation of mutant plasmid library

1. Amplification of mutant plasmid library

Due to the strong restriction modification in Streptomyces avermitilis, the transformation efficiency of Streptomyces avermitilis is very low when the plasmid extracted from E.coli DH5α is used to directly transform Streptomyces avermitilis, sometimes even no transformant can be obtained. However, the transformation efficiency was significantly improved by using the plasmid from the recipient strain E.coli ET12567 without restriction modification. Therefore, the constructed mutant plasmid library and the control plasmid were respectively transformed into E.coli ET12567 (pUZ8002) (the non-patent literature that recorded 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 the Institute of Microbiology, Chinese Academy of Sciences) to obtain unmethylated DNA, and then use unmethylated plasmid DNA Transformation of protoplasts from S. avermitilis.

The pZY126* expression plasmid vector library containing the mutated hrdB fragment constructed in Example 1 and the original plasmids pZY126 and pSET152 used as controls were electroporated to transform the competent cells of E.coli ET12567, and were coated with kanamycin, chloramphenicol and Apramycin LB plates were cultured at 37 degrees. After visible colonies grow on the plate, collect all the colonies with fresh liquid LB, add 15% glycerol and store at -80°C, or directly insert into LB containing corresponding antibiotics to further amplify the plasmid, extract the plasmid DNA, and digest verify.

2. Transformation of Streptomyces avermitilis by mutant plasmid library

In this example, Streptomyces avermitilis (Streptomyces avermitilis) ZLX6003 CGMCC 3229 was selected as the starting strain. ZLX6003 was preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: Beijing, China) on August 18, 2009. No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District). ZLX6003 is a high-yielding avermectin strain obtained through traditional breeding methods, which has been applied in industry and produces gray spores on plates.

Prepare the protoplasts of Streptomyces avermitilis strain ZLX6003, use each plasmid (pZY126*, pZY126 and pSET152) extracted from E.coliET12567 (pUZ8002) in step 1 to transform the protoplasts of the starting bacterial strain ZLX6003, apply to the dried Add antibiotics on the RM 14 plate, culture at 28°C for 16-20h, cover the plate with 1mL aqueous solution containing 1mg of apramycin, continue to culture at 28°C for 7-10 days, and the grown colony is the transformant. Randomly pick transformants with uniform colony morphology, inoculate on MS plates containing 20ug/mL apramycin, and culture at 28°C for 7 days to restore production. After the transformant was verified to be correct by plasmid extraction and PCR, the next step of fermentation research was carried out.

In this embodiment, the transformation of Escherichia coli, the preparation and transformation method of Streptomyces avermitilis protoplasts, and the preparation of RM 14 and MS medium refer to Zhang Xiaolin's doctoral thesis (Zhang Xiaolin. Genetic modification of polyketide synthase gene in Streptomyces avermitilis) .PhD Dissertation, 2004, Beijing: China Agricultural University).

Embodiment 4, the fermentation of transformant

1. Fermentation and yield analysis of ZLX6003 and its transformants

1. High-throughput screening of Streptomyces avermitilis

After the original strain ZLX6003 of Streptomyces avermitilis and its different transformants grow abundant spores on the slant medium, the abamectin production level of the Streptomyces avermitilis transformants was tested, and a high-throughput screening method was used for preliminary screening. Screening (Gao H, Liu M, Zhou X et al. Identification of avermectin-high-producing strains by high-throughput screening methods. Appl Microbiol Biotechnol, 2009, 85: 1219-1225.)

The strains with improved yield obtained from the primary screening were selected as shake flask fermentation for verification, and 1 cm ² of the slant lawn was excavated, inserted into a seed bottle containing 40 mL of sterilized seed medium, and cultured on a shaker at 28°C for 44-48 hours, the rotation speed is 200 rpm, and the rotation radius is 50 mm to obtain the seed culture solution. Get above-mentioned seed culture solution and inoculate in the Erlenmeyer flask that 30mL sterilized fermentation medium is housed by the inoculation amount of 5% (percentage by volume), 28 ℃ of shaker cultures 10 days, put bottle, measure Avermella with HPLC method The fermentation unit of element, specific HPLC analysis is as described in the following step 2.

For the preparation of the slant medium, seed medium and fermentation medium in this example, refer to the existing patent (No. 200810227639.8, a method for preparing abamectin and its special strain).

2. HPLC analysis of fermentation products

1) Sample treatment: take 1.0 mL of fermentation broth, add 9.0 mL of methanol, shake at a speed of 200 rpm (rotation radius is 50 mm) for 6 hours, and centrifuge at a speed of 8000 rpm for 5 min;

2) Take 1 mL of the supernatant, filter it with a 0.45um microporous membrane, and obtain the filtrate for HPLC analysis.

3) Take the fermentation filtrate in step 1, inject it into an autosampler, and the injection volume is 10ul;

Carry out separation with methanol: water (volume ratio is 9: 1) as mobile phase, and flow velocity is 1mL/min, utilizes UV detector to detect and form separation spectrum automatically at wavelength 245nm place; Under this chromatographic condition, Abamectin B1a The retention time of the standard (DR, Germany) is about 6 minutes. Calculate the retention time in the fermentation filtrate as the elution peak area at about 6 minutes, and calculate the amount of Abamectin B1a. The experiment was repeated 3 times, and the results were averaged.

The fermentation result of Fig. 3 shows, the avermectin production after the transformation of control plasmid pZY126 (represented by pSET152-hrdB among the figure) and blank control plasmid pSET152 starting strain ZLX6003 and the output of abamectin of starting bacterial strain ZLX6003 have no significance difference; and the output of the avermectin of the bacterial strain (containing the transformant of the mutant hrdB gene) that the above-mentioned output improves has increased by 52% compared with the output of the original bacterial strain ZLX6003, and this high-yielding bacterial strain is as engineering bacterium, is Streptomyces avermitilis (Streptomyces avermitilis) ZLX6056 CGMCC №.3796, has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on May 4, 2010, and the preservation number is CGMCCNo.3796.

2. Sequencing and sequence analysis of the mutant hrdB gene in the recombinant strain and verification of its impact on the production of abamectin

1. Sequencing and sequence analysis of the mutant hrdB gene

The genomic DNA of engineering bacteria ZLX6056 was extracted, digested completely with PstI, purified by ethanol precipitation, and then self-ligated. The self-ligated product was transformed into E. coli competent cells, and spread to LB containing apramycin resistance. Plates were cultured at 37°C until white colonies appeared. Pick a single colony and culture it to extract the plasmid DNA, digest it with XbaI and EcoRI and recover the 2.3kb mutant hrdB gene fragment including the promoter, connect the 2.3kb gene fragment with the vector pUC18 that has been digested with the same enzyme and transform it Escherichia coli DH5α competent cells were spread onto LB plates containing ampicillin resistance and cultured at 37°C until white colonies appeared. After picking a single colony and culturing it, the plasmid DNA was extracted, and after verification by enzyme digestion, it was sent to Beijing Huada Company for sequencing. Sequencing results showed that the nucleotide sequence of the 2.3 kb gene fragment was shown as sequence 5 in the sequence listing. Wherein the nucleotide sequence shown in the 658-2197th position from the 5' end of the sequence 5 is consistent with the sequence 3 in the sequence list, which is the sequence of the mutated hrdB gene, and can encode the mutated gene shown in the sequence 4 in the sequence list hrdB protein.

Sequence alignment was performed between the mutated HrdB protein shown in sequence 4 and the pre-mutated HrdB protein shown in sequence 2, and their conserved functional domains were analyzed. The results are shown in Figure 4, the full length of the protein sequence before and after the mutation is 512AA. The mutation sites were concentrated in regions 1.1 and 2.4. There are six point mutations in the mutated hrdB protein, namely A137S, K139E, E163G, M356V, V357A, and M389I. It is reported in the literature that the 2.4 region is mainly responsible for the combination of the promoter-10 region of the gene, and its stability is very high, while there are 2 point mutations in the engineering bacteria ZLX6056. The results further showed that the addition of a copy of the mutant hrdB gene in the engineering bacteria ZLX6056 affected the gene expression level of the biosynthesis-related pathways of abamectin, thereby increasing the production of abamectin.

2. Verification of the influence of the mutated hrdB fragment on the production of abamectin

1) Construction of recombinant expression vector

The mutated hrdB gene fragment containing the promoter of 2.3kb recovered in step 1 and the carrier pSET152 after XbaI and EcoRI double digestion (the non-patent literature that has recorded this material is Bierman M, Logan R, O'Brien K , et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43-49), the public can obtain from the Institute of Microbiology, Chinese Academy of Sciences) to obtain a recombinant expression vector. Then transform the recombinant expression vector into Escherichia coli E.coli ET12567 (the non-patent literature that recorded this material is MacNeil DJ, Gewain KM, Ruby CL, et al (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61-68., the public can obtain from the Institute of Microbiology, Chinese Academy of Sciences) Competent cells were spread on LB plates containing chloramphenicol, kanamycin and apramycin resistance, and cultured at 37°C until white colony. The plasmid DNA was extracted after picking a single colony for culture, and further sequenced after enzyme digestion verification, indicating that the obtained recombinant expression vector was formed by inserting the DNA fragment described in Sequence 5 in the sequence listing between the XbaI and EcoRI restriction sites of pSET152 .

2) transform Streptomyces avermitilis ZLX6003 bacterial strain and carry out fermentation

Transform the recombinant expression vector obtained in step 1) into the protoplasts of Streptomyces avermitilis ZLX6003 strain, apply it on the dried RM 14 plate without antibiotics, and after culturing at 28°C for 16-20h, cover the plate with 1mL containing The aqueous solution of 1 mg of apramycin was continued to culture at 28° C. for 7-10 days, and the grown colonies were transformants. Randomly pick transformants with uniform colony morphology, inoculate on MS plates containing 20ug/mL apramycin, and culture at 28°C for 7 days to restore production. . The correct transformants verified by PCR were named pZY148-hrdB ^ZLX6056 , and then 5 transformants verified by PCR were randomly selected and fermented by the method of Step 1 and Step 2 in Example 4.

At the same time, the vector pSET152 was transformed into the protoplast of Streptomyces avermitilis ZLX6003 strain to obtain an empty vector control strain.

Then, the empty vector control strain, the starting strain ZLX6003 and the engineering strain ZLX6056 were also subjected to the above-mentioned fermentation culture.

The fermentation experiment was repeated 3 times, and the results are shown in Table 2 (the B1a yield of the starting strain ZLX6003 after 240 hours of fermentation is defined as 100%). After 240 hours of fermentation, the recombinant strain pZY148-hrdB ^ZLX6056 can reach 148.23%, which can restore the engineering strain ZLX6056 The growth and yield characteristics of the empty vector control strain and the B1a yield of the starting strain ZLX6003 had no significant difference.

Therefore, it can be determined that the mutated hrdB gene shown in sequence 3 in the sequence listing can increase the production of abamectin.

Table 2. Functional verification of the mutated hrdB gene

Embodiment 5, engineering strain genetic stability detection

The engineering bacterial strain ZLX6056 of the present invention is carried out slant passaging, passes on altogether 5 generations, after 28 ℃ cultivates to grow vigorous spore (about 12～15 days), the bacterium obtained in each generation culture all according to embodiment 4 step-step 1 shake Bottle fermentation method is fermented, carries out fermentation and extraction by the method described in embodiment 4 step-step 2, and calculates the ability of bacterial strain production Abamectin B1a. The experiment was repeated 3 times, and the results were averaged. The results show that the production capacity of the engineering strain ZLX6056 of the present invention can still maintain the original level after 5 passages, indicating that the engineering strain ZLX6056 of the present invention has good genetic stability.

Embodiment 6, the fermentation enlargement of engineering bacterial strain

This embodiment compares the abamectin production capacity of the starting strain ZLX6003 and the engineering strain ZLX6056 containing the mutated hrdB gene on a 180-ton fermenter, and intermittent sampling during the fermentation process analyzes the changes in the avermectin output .

Wherein the fermentation steps are as follows:

Prepare the fermentation medium according to the formula in the feeding tank and adjust the pH, pump the prepared medium into the 180m ³ fermenter, after readjusting the pH, sterilize at 121℃～130℃ for 30 minutes to 1 hour, and then sterilize Cool to 28 degrees after completion, readjust the pH and set aside. For the seeds cultivated to the logarithmic phase, pump about 10m ³ (±20%) of the seed liquid into the above-mentioned sterilized fermenter under sterile conditions, start the fermentation and cultivation program, cultivate at 28-29°C, and control DO at About 50%, the pH is controlled at about 7.8, the stirring speed is 0-100rpm in the early stage, 100-200rpm after 24 hours, and about 200-300rpm after 72 hours. The growth of mycelia and the production of factors are regularly detected, and the fermenter is put into operation after about 312 hours.

See the existing patent (No. 200810227639.8, a method for preparing abamectin and its special strain) for the preparation of the seed medium and the fermentation medium in this example.

Result as shown in Figure 5, engineering strain ZLX6056 can still maintain the advantage of output growth after 200 hours on large-scale fermentor, and the ability of engineering strain of the present invention to produce abamectin B1a reaches 6382 μ g/ml fermentation in 312 hours solution, compared with the original strain ZLX6003 strain (4167μg/ml), the ability to produce 53.1% increased.

sequence listing

<110>Institute of Microbiology, Chinese Academy of Sciences

<120>A genetic engineering method for producing avermectin and its special strain

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gtgtcggcca gcacatcccg tacgctcccg ccggagatcg ccgagtccgt ctctgtcatg 60

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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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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gtgtcggcca gcacatcccg tacgctcccg ccggagatcg ccgagtccgt ctctgtcatg 60

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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

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gcgcccgcgc agcaggtcgc cgcggccggt gccaccgccg acccggtcaa ggactacctc 660

aagcagatcg gcaaggtccc cctgctcaac gccgagcagg aggtcgagct cgccaagcgc 720

atcgaggcgg gcctcttcgc cgaggacaag ctggccaacg ccgacaagct tgcccccaag 780

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cgcgccatgg ccgaccaggc ccgcaccatc cgtatcccgg tgcacgtggc cgaggtcatc 1080

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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

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

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

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

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

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

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<213> Streptomyces avermitilis

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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

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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 accacccggca 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 attacccc 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.

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