CN108753799A - A kind of mibemycin synthesis positive regulating gene kelR, coding albumen, genetic engineering bacterium and its preparation method and application - Google Patents

Abstract

A kind of mibemycin synthesis positive regulating gene kelR, coding albumen, genetic engineering bacterium and its preparation method and application, belong to field of microbial pharmacy, the present invention constructs the over-express vector containing the gene and upstream promoter sequence, and it is transformed into Escherichia coli, obtained transformant carries out Conjugal transfer with Harbin streptomycete, there is picking the joint element of apramycin resistance to be overexpressed genetic engineering bacterium to get to kelR.The present invention is suitable for the biosynthesis of mibemycin.

Description

A milbemycin synthesis positive regulatory gene kelR, encoded protein, genetically engineered bacteria And its preparation method and application

technical field

The invention belongs to the field of microbial pharmacy, and in particular relates to a milbemycin synthesis positive regulation gene kelR, a protein encoded by the gene, a genetically engineered bacterium overexpressing kelR, a preparation method and application thereof.

Background technique

Biopharmaceuticals, especially macrolides, are being and are increasingly being used in veterinary medicine and agriculture. Milbemycin is one of the representative commercialized pesticides and veterinary drugs with broad-spectrum inhibition of pests and good eco-friendliness. Milbemycin is a class of sixteen-membered macrolide antibiotics with a chemical structure similar to abamectin. Milbemycin was first discovered in Japan in 1967, and was first registered as an acaricide in Japan for pest control of crops and commercial crops. It was then registered in Australia in 2006 as an acaricide against the flower-concentrating Tetranychus urticae. Up to now, milbemycin has been approved in 43 countries and regions including the United States, China, and Italy for the control of pests in 24 crops, cash crops and flowers. Milbemycin (A3:A4=30:70) has a slightly higher acaricidal activity than Avermectin, the most highly effective acaricide, and is 40 times less toxic to rats than Avermectin. It is therefore considered a less hazardous pesticide by the US EPA. The Netherlands has approved it as a natural product in the crop production process, and it is an eco-friendly pesticide. Milbemycin has also become a very popular acaricide in many countries such as the United States and Canada, and has a good application prospect. Some derivatives of milbemycin also exhibit very good biological activity and have been applied commercially. For example, milbemycin is a semi-synthetic product of milbemycin, which is a 5-oxime derivative of milbemycin A3 and A4 (A3:A4=20:80), and has anti-microfilaria activity. Safe antiparasitic drug. It is commonly used to prevent dirofilariasis and to treat diseases in cats and dogs caused by hookworms and nematodes, as well as to control whipworms in dogs. The sales volume of milbexime ranks second in the antiparasitic drugs in the European and American markets, and the application prospect is very good. In addition, Lepimectin, a semi-synthetic drug of milbemycin, has been registered for the control of pests such as Lepidoptera and Hemiptera in vegetables and fruits. For the treatment of human skin diseases; semi-synthetic Latidectin (Latidectin) has also been commercialized for the control of animal parasites.

Although the milbemycin series products have huge commercial value, countries all over the world are also scrambling to develop and produce milbemycin strains, but they have not succeeded. In the past half a century, only Japan Sankyo Company has monopolized the production of milbemycin technical products. . In 1999, the research group screened out a new milbemycin-producing strain (4 authorized invention patents: CN101100651B, CN10046743C, CN100467472C, CN100490648C), which was named Streptomyces bingchengensis. This strain provides the basis for the industrial production of milbemycin in my country. In addition to the known milbemycins, Streptomyces glaciensis also produces 12 unreported milbemycins, cyclic pentapeptides, a macrolide compound and polyether insecticides Nanchang Mycin.

Streptomyces iceberg completed the whole genome sequencing in 2010, the GeneBank accession number is CP002047, and its genome size is 11,936,683bp. There are at least 47 gene clusters related to the biosynthesis of compounds such as polyketides, non-ribosomal peptides or terpenoids in the genome, including the milbemycin biosynthesis gene cluster, so that the biosynthesis gene cluster of milbemycin The structure, distribution and biosynthesis process of milbemycin-producing bacteria have a better understanding (Wang X et al. Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis. JBacteriol. 2010; 192: 4526-4527). There are four main open reading frames (milA1, milA2, milA3, milA4) in the milbemycin biosynthetic gene cluster in Streptomyces icebergi, which encode 12 modules responsible for the polyketide activity of milbemycin synthesis. There is only one positive regulatory gene milR in the milbemycin biosynthesis gene cluster. It has been confirmed that milR controls the biosynthesis of milbemycin by directly regulating the transcription of milA4 and milF, but directly regulates the regulation of milA1, milA2 and milA3. The gene is still not found (Zhang Y et al. Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microbialcell factories. 2016; 15, 152).

Contents of the invention

Aiming at the problem of how to determine the regulatory genes that directly regulate milA1, milA2 and milA3 in the milbemycin biosynthesis gene cluster, the present invention provides a milbemycin synthesis positive regulatory gene kelR, the nucleotide sequence of the kelR gene As shown in SEQ ID No.1.

The invention provides a positive regulatory protein KelR for milbemycin synthesis, which is encoded by the positive regulatory gene kelR for milbemycin synthesis according to claim 1. The KelR protein consists of 274 amino acids, and its amino acid The sequence is shown as SEQ ID No.2.

The present invention also provides a high-yield milbemycin genetically engineered bacterium, said genetically engineered bacterium overexpresses the positive regulatory gene kelR for milbemycin synthesis according to claim 1, and its nucleotide sequence is as shown in SEQ ID No.1 As shown, the starting strain is one of Streptomyces bingchenggensis, Streptomyces milbemycin, Streptomyces hygroscopicus subsp.

Preferably, the Streptomyces bingchenggensis is Streptomyces bingchenggensis BCWT or Streptomyces bingchenggensis BC-120-4.

The preparation method of the above-mentioned high-yielding milbemycin genetically engineered bacteria comprises the following steps:

1) Construct the overexpression vector of milbemycin synthesis positive regulation gene kelR, the overexpression vector contains milbemycin synthesis positive regulation gene kelR, its sequence is shown in SEQ ID No.1; and milbemycin synthesis positive Regulatory gene kelR upstream promoter, its sequence is shown in SEQ ID No.3;

2) Transforming the Escherichia coli host cell with the milbemycin synthesis positive regulation gene kelR overexpression vector constructed in step 1), to obtain a transformant containing the milbemycin synthesis positive regulation gene kelR overexpression vector;

3) Intergenus conjugation and transfer of the transformant obtained in step 2) with Streptomyces to obtain a high-yield milbemycin genetically engineered bacterium.

Preferably, the construction process of the overexpression vector of the milbemycin synthesis positive regulatory gene kelR described in step 1) is:

Using the Streptomyces glaciferi genome as a template, PCR amplifies the gene fragment containing the complete coding region of the milbemycin synthesis positive regulatory gene kelR and the upstream promoter, and the upstream primer sequence used for the PCR amplification is as SEQ ID No.4 As shown, the sequence of the downstream primer is shown in SEQ ID No.5; the restriction endonuclease XbaI and EcoRI are used to digest, and the obtained digestion product is connected to the pSET152 vector that has been digested with XbaI and EcoRI to obtain the recombinant plasmid pSET152::kelR is the overexpression plasmid of the milbemycin synthesis positive regulatory gene kelR; the Streptomyces bingchenggensis is Streptomyces bingchenggensis BCWT or Streptomyces bingchenggensis BC-120-4.

Preferably, the Streptomyces in step 3) is one of Streptomyces bingchenggensis BCWT, Streptomyces bingchenggensis BC-120-4, Streptomyces milbemycin, Streptomyces hygroscopicus subsp. aureus, and Streptomyces griseochromogenes.

The Streptomyces bingchenggensis BCWT is described in Gao Aili, Wang Xiangjing, et al. (2007). "Screening and Identification of New Species of Streptomyces hygroscopicus." Journal of Northeast Agricultural University 38(3):361-364.

The Streptomyces bingchenggensis BC-120-4 described in "Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis" was described in Wang, H.Y., J. Zhang, et al. ."Applied Microbiology and Biotechnology 98(23):9703-9712.

Other strains are recorded in Lin Xiuping, Liu Yonghong, Li Jilun. Research progress on producing bacteria, physical and chemical properties, biological activity and application of milbemycin[J]. Chinese Journal of Antibiotics, 2013,38(04):314-322.

Preferably, the Escherichia coli host cell described in step 2) is E. coli ET12567\pUZ8002.

The milbemycin synthesis positive regulatory gene kelR of the present invention can increase the yield of milbemycin in microbial fermentation production of milbemycin.

The high-yielding milbemycin genetically engineered bacteria can be used in fermentative production of milbemycin, cultivating high-yielding milbemycin genetically engineered strains, and isolating milbemycin from the culture.

Those skilled in the art will understand that if the kelR gene shown in SEQ ID No.1 is mutated by one or more nucleotides, such as substitution, addition, deletion, etc., if it can still encode The activity of protein synthesis, even when compared to the enhanced activity of the protein shown in SEQ ID No. 2, is still applicable to the present invention.

Those skilled in the art will understand that if the KelR protein shown in SEQ ID No.2 is mutated and/or modified by one or more amino acids, such as substitutions, additions, deletions, etc. The activity of protein synthesis, even when compared to the enhanced activity of the protein shown in SEQ ID No. 2, is still applicable to the present invention. The gene encoding the polypeptide can also be used in the present invention to construct an engineering bacterium that improves the production of milbemycin.

Beneficial effect

The present invention discovers for the first time the function of the positive regulatory gene kelR which directly regulates the synthesis of milbemycin and its encoded protein. This protein positively regulates the synthesis of milbemycin. The positive regulatory gene kelR for the synthesis of milbemycin corresponds to the gene SBI_06842 (GeneBank accession number) in Streptomyces iceberg. The research of the present invention shows that the deletion of kelR directly leads to the termination of the synthesis of milbemycin, and reintroduces a The copied kelR restores the synthesis of milbemycin, and overexpression of kelR can promote the production of milbemycin. It indicated that the regulatory protein encoded by the kelR gene was a positive regulatory protein of the milbemycin synthesis gene cluster.

The function of kelR was investigated by homologous recombination double crossover, and it was confirmed that it can positively regulate the biosynthesis of milbemycin and plays a decisive role in the synthesis of milbemycin.

On this basis, the present invention has constructed a high-yielding milbemycin genetically engineered bacterium. By increasing the copy number of the kelR gene in Streptomyces iceberg, overexpressing kelR can promote the production of milbemycin. Therefore, the production cost is reduced and the production cost is improved. economic benefit.

Description of drawings

In Figure 1, a is the kanamycin resistance gene plasmid pUC119::neo; b is the plasmid pKC1139 for gene blocking; c is the physical spectrum of the plasmid pSET152 for overexpression.

Figure 2 is a schematic diagram of the construction of kelR blocking mutants and anaplerotic mutants, WT is wild type.

Fig. 3 is a spectrogram of HPLC analysis of the fermentation broth of the kelR blocking mutant strain and its anaplerizing mutant strain; the abscissa is the retention time in minutes, and the ordinate is the absorbance in mAu.

Fig. 4 is a schematic diagram of construction of kelR gene overexpression strain.

Detailed ways

The present invention will be further described below by way of examples, but the present invention is not limited thereto. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

The vector pUC119::neo of the present invention was purchased from Protin Biotechnology (Beijing) Co., Ltd.

The plasmid miniprep kit was purchased from AxyPrep, catalog number: AP-MN-P-250G.

The gel recovery kit was purchased from AxyPrep DNA, catalog number: AP-GX-250.

The vector pKC1139 was purchased from BioVector Plasmid Vector Strain Cell Gene Collection Center.

The formula of LB medium is: tryptone 10g, yeast extract powder 5g, sodium chloride 10g, distilled water 1L, pH 7.2-7.5. Sterilize at 121℃, 1.01×105Pa for 20 minutes.

The formula of 2×YT medium is: tryptone 16g, yeast extract powder 10g, sodium chloride 5g, distilled water 1L, pH 7.2-7.5. Sterilize at 121°C and 1.01×105Pa for 20 minutes.

Example 1. Research on the function of milbemycin synthesis positively regulating gene kelR.

1. Construction of milbemycin synthesis positive regulatory gene kelR blocking mutant strain

Using primers 6842up-f/6842up-r from Streptomyces bingchenggensis BCWT (the Streptomyces bingchenggensis BCWT is recorded in Gao Aili, Wang Xiangjing, et al. (2007). "Screening and identification of new species of Streptomyces hygroscopicus." Northeast Agriculture University Journal 38(3):361-364.) PCR amplification on the genome obtained the homologous recombination left arm fragment of the kelR gene, ie the upstream fragment of kelR.

Using primers 6842down-f/6842down-r, the right arm fragment of kelR gene homologous recombination, namely the downstream fragment of kelR, was obtained by PCR amplification from Streptomyces bingchenggensis BCWT genome.

The Streptomyces bingchenggensis BCWT genomic DNA was extracted according to the method described in Streptomyces genetics manual (Kieser, T., M.J. Bibb, et al. (2000). Practical streptomyces genetics, John Innes Foundation Norwich). .

The primer sequence was synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (the same below), and the underline represents the restriction endonuclease cutting site (the same below):

6842up-f: 5' CCC AAGCTT GTGGCGTGGAAGCAGTTGAGGAAGGC 3'

( AAGCTT: HindIII restriction site)

6842up-r: 5'GC TCTAGA CACCGGGAGCATCTGGTTGGCGTA 3'

( TCTAGA: XbaI restriction site)

6842down-f: 5'CG GAATTC GACGATGTCGAGGCTTCGTTTCTGTTCC 3'

( GAATTC: EcoRI restriction site)

6842down-r: 5'GC TCTAGA TACTCGGTGTCCAGGCTGGTGGTGG 3'

( TCTAGA: XbaI restriction site)

PCR reaction system: 5 μL of 10×KOD-Plus buffer, 5 μL of 2mM dNTPs, 2 μL of 25mM MgSO ₄ , 1 μL of KOD-Plus (1U/μL) (the above reagents were purchased from TOYOBO, Japan); 10 μM primers 6842up-f and 6842up-r 1.5 μL each, (or 1.5 μL each of 10 μM primers 6842down-f and 6842down-r), genome template 1 μL (1-50 ng), 32 μL, DMSO 2.5 μL, add ddH ₂ O to 50 μL.

PCR cycle conditions: pre-denaturation: 94°C, 2min. Denaturation: 94°C, 15sec; Annealing: 60°C, 30sec. Extension: 68°C, 1min, 30 cycles; 68°C, 2min, 4°C, storage.

The upstream fragment 1875bp and the downstream fragment 2076bp of kelR were amplified. The upstream fragment was double-digested with HindIII and XbaI (restriction enzymes were purchased from TAKARA, the same below), and the downstream fragment was double-digested with XbaI and EcoRI. Agarose DNA Recovery Kit for recovery. The upstream fragment was ligated into the backbone of plasmid pUC119::neo digested with HindIII and XbaI (the physical spectrum of the plasmid is shown in Figure 1 a), and the resulting recombinant plasmid was pUC6842upneo. Digest pUC6842upneo with HindIII and EcoRI, recover the 2874bp upneo fragment, and connect the digested product of the upneo fragment and the 2076bp downstream fragment of kelR into pKC1139 digested with HindIII and XbaI (the physical spectrum of the plasmid is shown in b in Figure 1 Shown, pKC1139), to obtain kelR blocking plasmid pKC6842. The kelR blocking plasmid pKC6842 was transformed into E.coli ET12567\pUZ8002 competent cells, and then transferred into Streptomyces icebergi BCWT by indirect conjugation transfer. Conjugates with resistance to Apramycin (Apr) or Kanamycin (Kanamycin, Kan) were screened. The zygote obtained is transferred to culture in the liquid medium SSPY containing kanamycin and apramycin, and the final concentration of the kanamycin in the SSPY medium is 25ug/ml, and the apramycin The final concentration of antibiotics in the SSPY medium is 30ug/ml. For other mediums involving kanamycin and apramycin in this application, the concentration of antibiotics is added according to this concentration. Pick 6 of the zygotes verified by plasmid extraction and enzyme digestion and transfer them to SSPY plates containing kanamycin, culture them at 28°C for 7 days, prepare spore suspensions, and spread them at a concentration of about 104 spores per plate ^. Spread on SSPY plates containing kanamycin and culture at 37°C. The plasmid extractions described in this application were all carried out using the AxyPrep plasmid miniprep kit.

pKC1139 has a temperature-sensitive replicon, which cannot replicate autonomously when cultured at a temperature higher than 34°C. Only when the recombinant plasmid undergoes homologous recombination with the chromosomal DNA of Streptomyces icebergi can it grow on MS medium containing kanamycin. If a double crossover occurs, the kanamycin resistance gene is correctly inserted into the target gene, and the colony shows Kan ^R Apr ^S (Kanamycin resistance, Apramycin sensitivity). Copy the colonies expressing Kan ^R Apr ^S onto SSPY/Kan and SSPY/Apr plates at the same time, and culture them at 28°C. Mutant strains were randomly selected, inoculated on SSPY medium without any selection pressure, and cultured at 28°C. After three generations of transfer, re-inoculated on SSPY medium containing kanamycin and apramycin respectively, the results still showed Kan ^R Apr ^S , indicating that a genetically stable kelR blocking mutant strain was obtained through double crossover, named ΔkelR, see ΔkelR shown in the second row of Fig. 2 .

The genome of the kelR blocking mutant was extracted according to the method described in the Streptomyces Genetic Operation Manual, and PCR amplification was performed using primers con6842-f/con6842-r for identification.

con6842-f: 5'TGCTCGTCGAACGGATCGCAGACC 3'

con6842-r: 5'TGGAACGACAGGACCCACGGAACG 3'

The PCR reaction system and cycle conditions are the same as above. After double crossover, using the genomic DNA of the kelR blocking mutant strain ΔkelR as a template, the theoretical size of the fragment amplified with primers con6842-f/con6842-r is 3902bp, and the fragment size amplified using the genome of the starting strain BCWT as a template It is 3144bp.

2. Construction of milbemycin synthesis positive regulatory gene kelR anaplerotic mutant strain

Using the Streptomyces bingchenggensis BCWT genome as a template, primers 6842self-f/6842self-r were used to amplify the regulatory gene kelR and its promoter region with a total of 1653bp, and the corresponding nucleotide sequence is shown in SEQ ID No.6.

6842 self-f: 5'GC TCTAGA GGTGCGGGGAGAAGCCCAATG 3'

( TCTAGA: XbaI restriction site)

6842self-r: 5'G GAATTC CCGACAGGACCCACGGAACG 3'

( GAATTC: EcoRI restriction site)

PCR reaction system: 10×KOD Buffer 5 μL, dNTPs (2mM) 5 μL, template DNA (Streptomyces iceberg BCWT genome) 10pg-1000ng, 10 μM upstream primer 6842self-f, downstream primer 6842self-r (shown in SEQ ID No.4 , shown in SEQ ID No.5; 10 μM) 2 μL each, 25 mM MgSO ₄ 2 μL, KOD plus DNA polymerase (1 U/μL) 1 μL, DMSO 2.5 μL, ddH ₂ O to 50 μL. Reaction conditions: 94°C for 4min, 94°C for 1min, 65°C for 30sec, 68°C for 3min, 31 cycles, 68°C for 10min. For the recovery of 1653bp PCR products by gel cutting, the gel recovery described in this application was carried out using the AxyPrep DNA Gel Recovery Kit. Use XbaI and EcoRI endonucleases to double-enzyme-digest the above gel recovery product and vector pSET152 respectively. The size of the double-digestion product of the gel recovery product is 1651bp, and the size of the double-digestion product of pSET152 is 5685bp:

Digestion system: kelR fragment or vector pSET152 1-2 μg, 10×M buffer 10 μL, XbaI 2.5 μL, EcoRI 2.5 μL, ddH ₂ O to 100 μL) (see c in Figure 1 for the physical spectrum of pSET152 plasmid). (The pSET152 plasmid is described in Bierman M et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992; 116:43-9).

Then the digested products were ligated:

The ligation system: vector pSET152 10-20ng, kelR fragment 50-100ng, 10×T ₄ DNA ligase buffer 1 μL, T ₄ DNA ligase 0.8 μL, ddH ₂ O to make up to 10 μL.

Transform the above ligation product into Escherichia coli DH5α after 3 hours in a water bath at 22°C, pick a single clone after culturing overnight on an LB medium plate at 37°C, and culture overnight in LB medium in a small test tube at 37°C, and extract the plasmid The recombinant plasmid pSET152::kelR was obtained, and the vector map is shown in FIG. 4 . The pSET152::kelR was transformed into E.coli ET12567\pUZ8002 competent cells, and transferred into the kelR blocking mutant strain by intergenus conjugation.

The transformants were screened with the apramycin resistance marker, pSET152 is an integrated vector, which cannot replicate autonomously in Streptomyces, and can only undergo specific recombination between the ФC31attP site of the phage carried on the pSET152 vector and the attB site of the Streptomyces genome The anaplerotic mutant ΔkelR::kelR is integrated into the chromosome and combined with the chromosome to obtain the kelR blocking mutant, as shown in the third row of FIG. 2 ΔkelR::kelR.

Example 2. Analysis of milbemycin synthesis of milbemycin synthesis positive regulatory gene kelR blocking mutant strain and anaplerotic mutant strain.

1. Fermentation culture adopts the following medium

Seed medium (1L): 10g sucrose, 1g skimmed milk powder, 3.5g peptone, 5g yeast extract powder, 0.5g K ₂ HPO ₄ 3H ₂ O, 1L distilled water, pH 7.0. Sterilized at 121°C, 1.01×10 ⁵ Pa 20 minutes.

Fermentation medium (1L): 80g sucrose, 20g soybean cake powder, 1g skimmed milk powder, 1g K ₂ HPO ₄ 3H ₂ O, 0.1g FeSO ₄ 7H ₂ O, 3g CaCO ₃ , 1L distilled water, pH 7.0-7.2. Sterilize at 121°C, 1.01×10 ⁵ Pa for 30 minutes.

2. Fermentation process

The pre-prepared spores of Streptomyces bingchenggensis BCWT, the knockout mutant strain ΔkelR constructed above and the complementation mutant strain ΔkelR::kelR were respectively streaked and activated on the MS plate, and about 2 cm ² mycelium pieces were scooped out Inoculate into a Erlenmeyer flask containing 25 mL of the above-mentioned seed medium, and cultivate for 2 days at 28° C. with shaking at 250 r/min. The obtained seed bacteria solution was transferred to the fermentation medium according to the inoculum amount of 5% (volume fraction), and cultured with shaking at 28° C. and 250 r/min for 10 days.

3. HPLC analysis of milbemycin in fermentation broth

Take 0.5 mL of fermentation feed liquid, add 1.5 mL of absolute ethanol, shake for 15 min, and then centrifuge at 4000 rpm for 15 min. After the supernatant was filtered with a 0.22 μm membrane filter, the filtrate was stored in a -20°C refrigerator.

HPLC assay conditions are as follows:

Chromatographic column: Agilent TC-C18, 5 μm, 150×4.6mm.

Mobile phase: A: MeCN-H2O-MeOH (350:50:100, v/v/v), B: MeOH.

Flow rate: 1 mL/min.

Detection wavelength: 242nm.

Injection volume: 10 μL.

Analysis: 0-15min, phase B gradient elution from 0% to 100%.

As shown in Figure 3, using Streptomyces icebergi wild-type BCWT fermentation broth as a control, it was found that the kelR blocking mutant strain ΔkelR milbemycin synthesis was terminated, that is, no production of milbemycin could be detected; while the complementation mutation Strain ΔkelR::kelR can resume the synthesis of milbemycin, indicating that kelR is a positive regulatory gene in the synthesis pathway of milbemycin, and is a switch that determines the synthesis of milbemycin.

Example 3. Construction of high-yielding milbemycin genetically engineered bacteria.

Transform the recombinant vector pSET152::kelR used in Example 1 into E.coli ET12567\pUZ8002 competent cells to obtain E.coli ET12567\pUZ8002 transformants containing pSET152::kelR, and transfer pSET152 :: kelR is transferred into Streptomyces bingchenggensis BC-120-4 bacterial strain (the BC-120-4 bacterial strain is recorded in Wang H et al. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4as main components fromStreptomyces bingchenggensis.Applied microbiology and biotechnology.2014;98:9703-9712), the specific process is:

The competent state of Escherichia coli ET12567\pUZ8002 was prepared, and the preparation method was described in the Streptomyces Genetic Operation Manual. The constructed plasmid was transformed into competent cells of ET12567\pUZ8002; spread on the LB medium plate containing Apr antibiotics, cultivated overnight at 37°C, picked a single colony of the transformant on the LB plate and transferred it to 4mL containing In the LB small test tube of Apr antibiotics, cultivate overnight at 37°C; inoculate the bacterial solution in the small test tubes into the Erlenmeyer flask containing Apr antibiotics at a volume ratio of 1:50, and cultivate at 37°C and 250rpm until the OD ₆₀₀ is about 0.4 Centrifuge at 4000rpm for 10min, discard the supernatant and collect the bacteria, wash twice with equal volume of LB liquid medium, collect the bacteria by centrifuging at 6000rpm for 10min, resuspend the precipitated bacteria in 0.1 times the volume of LB liquid medium. At the same time, prepare the spore suspension of Streptomyces BC-120-4, scrape the mature spores into a glass bead-triangle flask, and disperse the spores cultured in 5 5-15ml slant medium with about 20mL water, 250rpm, 30min shaking; Centrifuge at 4000rpm for 10min, discard the supernatant to recover the spore precipitate, add an equal volume of 2×YT liquid medium to wash once, collect the spore precipitate, and then resuspend in 5mL 2×YT liquid medium; after heat shock at 45°C for 10 minutes, Incubate at 28°C and 250rpm for 2 hours; mix the E.coli and Streptomyces iceberg BC-120-4 spore suspensions containing the recombinant vector pSET152::kelR, centrifuge at 3000rpm for 10min, discard the supernatant, and resuspend the pellet with the residue , smeared on the MS plate with a final concentration of 10mM MgCl ₂ ; cultured at 28°C for 20-24h, sterilized with 1mL ddH containing 1.5mg nalidixic acid and a final concentration of 4-6μg/mL apramycin ₂ O coating; after air-drying, continue to culture at 28°C for 4-5 days, and pick the zygotes with apramycin resistance to obtain high-yielding milbemycin genetically engineered bacteria.

Select 8 strains of engineering bacteria therefrom, carry out fermentation and Milbemycin liquid chromatographic detection to it according to the method for embodiment 2, compare with starting bacterial strain Streptomyces bingchenggensis BC-120-4, the result shows high-yield Milbemyces The average yield of milbemycin from the genetically engineered bacteria reached 4676mg/L, and the yield of milbemycin from the starting strain Streptomyces bingchenggensis BC-120-4 was 3833mg/L, an increase of about 22%. It shows that the kelR overexpression genetically engineered bacteria can obtain higher milbemycin production than the original strain.

Example 4. The difference from Example 1 is that the genome template used in this example is to replace Streptomyces bingchenggensis BCWT with Streptomyces bingchenggensis BC-120-4. Other steps are with embodiment 1.

The obtained milbemycin synthesis positive regulatory gene kelR blocking mutant and anaplerotic mutant were analyzed by milbemycin synthesis, indicating that kelR from Streptomyces bingchenggensis BC-120-4 was synthesized by milbemycin The positively regulated gene in the pathway is the switch that determines the synthesis of milbemycin.

Embodiment 5. The difference with Example 3 is: the starting strain Streptomyces bingchenggensis BC-120-4 of Milbemycin genetically engineered bacteria is replaced with any suitable bacterial strain producing Milbemycin, such as Milbemycin Streptomyces streptomyces, Streptomyces iceberg, Streptomyces hygroscopicus such as Streptomyces hygroscopicus subsp. aureus and Streptomyces griseus chromogenes, etc., other steps are the same as in Example 3, and finally high-yielding milbemycin genetically engineered strains are obtained.

Any suitable strains for the production of milbemycin are recorded in Lin Xiuping, Liu Yonghong, Li Jilun. Milbemycin producing bacteria, physical and chemical properties, biological activity and application research progress [J]. Chinese Journal of Antibiotics, 2013, 38(04):314-322.

SEQUENCE LISTING

<110> Institute of Plant Protection, Chinese Academy of Agricultural Sciences

<120> A milbemycin synthesis positive regulatory gene kelR, encoded protein, genetically engineered bacterium and its preparation method and application

<130>

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 825

<212>DNA

<213> kelR gene

<400> 1

atggaaatca aggtgttggg gccgctgagc gtcgaggcct gcggtcagtc gatcgtgccc 60

agcgcgggca agccgcggca gatcctcgca ttgctttccg tgtacgccaa ccagatgctc 120

ccggtgccca cccttatgga ggaaatctgg ggcgcgcaga tgccgcgcag tgcgttgacc 180

acgctccaga cgtacatcct tcagctccgg cggagactgg ccacggccta cggtccgcag 240

gcgcccgagg catcgaagag cgtgctggct acgcgatacg gcggatatgt gctggaggcc 300

cagccgggcg cggtggacat ccacgagtac gaccggctgg tggccgccgg gcgggacgcg 360

ctcgacgtcg gcgacgatgt cgaggcttcg tttctgttcc gcgaggcgct ggcggtctgg 420

cgcggtcccg cgctcgtgga cgtacgcgtc ggaccgatcc tggagatcga gctggcccgg 480

ctggaggaga gccgtctcgg ggtcctggag cgtcggatcg aggccgatct gcgcctggga 540

cgccacgccg aactcctcac cgagctcacc gaactgacgg cccggcatcc gctccacgag 600

gggctgcacg cccagtgcat ggctgccctg taccggtccg gccgcccatg gcaggcgctg 660

gaggtgtacc aggggctgcg cggccggctc gtcgaggacc tggggctcga accctccccg 720

cggctgcaga agctgcagca ggcggtgctc gcctcggacc ccgcgctcga cctggagtcg 780

ggcgggcagt ggcgccgccc ggtgctcgac ctgttcgccg cgtag 825

<210> 2

<211> 274

<212> PRT

<213> Amino acid sequence of KelR protein

<400> 2

Met Glu Ile Lys Val Leu Gly Pro Leu Ser Val Glu Ala Cys Gly Gln

1 5 10 15

Ser Ile Val Pro Ser Ala Gly Lys Pro Arg Gln Ile Leu Ala Leu Leu

20 25 30

Ser Val Tyr Ala Asn Gln Met Leu Pro Val Pro Thr Leu Met Glu Glu

35 40 45

Ile Trp Gly Ala Gln Met Pro Arg Ser Ala Leu Thr Thr Leu Gln Thr

50 55 60

Tyr Ile Leu Gln Leu Arg Arg Arg Leu Ala Thr Ala Tyr Gly Pro Gln

65 70 75 80

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

85 90 95

Val Leu Glu Ala Gln Pro Gly Ala Val Asp Ile His Glu Tyr Asp Arg

100 105 110

Leu Val Ala Ala Gly Arg Asp Ala Leu Asp Val Gly Asp Asp Val Glu

115 120 125

Ala Ser Phe Leu Phe Arg Glu Ala Leu Ala Val Trp Arg Gly Pro Ala

130 135 140

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

145 150 155 160

Leu Glu Glu Ser Arg Leu Gly Val Leu Glu Arg Arg Ile Glu Ala Asp

165 170 175

Leu Arg Leu Gly Arg His Ala Glu Leu Leu Thr Glu Leu Thr Glu Leu

180 185 190

Thr Ala Arg His Pro Leu His Glu Gly Leu His Ala Gln Cys Met Ala

195 200 205

Ala Leu Tyr Arg Ser Gly Arg Pro Trp Gln Ala Leu Glu Val Tyr Gln

210 215 220

Gly Leu Arg Gly Arg Leu Val Glu Asp Leu Gly Leu Glu Pro Ser Pro

225 230 235 240

Arg Leu Gln Lys Leu Gln Gln Ala Val Leu Ala Ser Asp Pro Ala Leu

245 250 255

Asp Leu Glu Ser Gly Gly Gln Trp Arg Arg Pro Val Leu Asp Leu Phe

260 265 270

Ala Ala

<210> 3

<211>611

<212>DNA

<213> Gene kelR upstream promoter

<400> 3

gaggtgcggg gagaagccca atgtgcccat cagccgacgc tgatggtgca gatgcagatg 60

cccggcgcgg gagcgggcga cggctcggcg gagaggtcct cgatgaccaa gtcgtcgacg 120

cccaagcggc ctcgggggagg tggggtgcgt tcccgcgtcg tccggtgcgg tgcatcgcaa 180

ggcggagcgg cgccctcgta ctgggcgtac tcgggtgctg cgacaacgcg gcgaggtgcc 240

gtgccgggcg gcgcgggggc gtgcctcact tccccgaggt cgcttgggtc ggcggggccg 300

gggcccgcgg cgcagagggc cgaagggctc gcggggagcg gggatgtgtc ggtcattgtg 360

ggctctcctc ggggtgagag gcggtcctgg caaggagtct gcgatgacgg atccacggct 420

tgccagtgct ttcctcataa ccagggtgtg aacttgtcat gacctccgcc tcacaggact 480

catgcacggt tcgggagtcc ggcactggcc gtgtccacca cccgcacgcg aggctcatat 540

gtgcctcaat ggcagggccg cgcggccaaa gaggcacctg gtggaccgtc tgacggaggt 600

gtgacaggac c 611

<210> 4

<211> 29

<212>DNA

<213> Upstream primer 6842self-f

<400> 4

gctctagagg tgcggggaga agcccaatg 29

<210> 5

<211> 27

<212>DNA

<213> downstream primer 6842self-r

<400> 5

ggaattcccg acaggaccca cggaacg 27

<210> 6

<211> 1653

<212>DNA

<213> Gene kelR and its promoter region

<400> 6

gctctagagg tgcggggaga agcccaatgt gcccatcagc cgacgctgat ggtgcagatg 60

cagatgcccg gcgcgggagc gggcgacggc tcggcggaga ggtcctcgat gaccaagtcg 120

tcgacgccca agcggcctcg gggaggtggg gtgcgttccc gcgtcgtccg gtgcggtgca 180

tcgcaaggcg gagcggcgcc ctcgtactgg gcgtactcgg gtgctgcgac aacgcggcga 240

ggtgccgtgc cgggcggcgc gggggcgtgc ctcacttccc cgaggtcgct tgggtcggcg 300

gggccggggc ccgcggcgca gagggccgaa gggctcgcgg ggagcgggga tgtgtcggtc 360

attgtggggct ctcctcgggg tgagaggcgg tcctggcaag gagtctgcga tgacggatcc 420

acggcttgcc agtgctttcc tcataaccag ggtgtgaact tgtcatgacc tccgcctcac 480

aggactcatg cacggttcgg gagtccggca ctggccgtgt ccaccacccg cacgcgaggc 540

tcatatgtgc ctcaatggca gggccgcgcg gccaaagagg cacctggtgg accgtctgac 600

ggaggtgtga caggaccatg gaaatcaagg tgttggggcc gctgagcgtc gaggcctgcg 660

gtcagtcgat cgtgcccagc gcgggcaagc cgcggcagat cctcgcattg ctttccgtgt 720

acgccaacca gatgctcccg gtgccccaccc ttatggagga aatctggggc gcgcagatgc 780

cgcgcagtgc gttgaccacg ctccagacgt acatccttca gctccggcgg agactggcca 840

cggcctacgg tccgcaggcg cccgaggcat cgaagagcgt gctggctacg cgatacggcg 900

gatatgtgct ggaggcccag ccgggcgcgg tggacatcca cgagtacgac cggctggtgg 960

ccgccgggcg ggacgcgctc gacgtcggcg acgatgtcga ggcttcgttt ctgttccgcg 1020

aggcgctggc ggtctggcgc ggtcccgcgc tcgtggacgt acgcgtcgga ccgatcctgg 1080

agatcgagct ggcccggctg gaggagagcc gtctcggggt cctggagcgt cggatcgagg 1140

ccgatctgcg cctgggacgc cacgccgaac tcctcaccga gctcaccgaa ctgacggccc 1200

ggcatccgct ccacgagggg ctgcacgccc agtgcatggc tgccctgtac cggtccggcc 1260

gcccatggca ggcgctggag gtgtaccagg ggctgcgcgg ccggctcgtc gaggacctgg 1320

ggctcgaacc ctccccgcgg ctgcagaagc tgcagcaggc ggtgctcgcc tcggaccccg 1380

cgctcgacct ggagtcgggc gggcagtggc gccgcccggt gctcgacctg ttcgccgcgt 1440

aggtccctgc cggtcccgct cggggctcag gcccttctgc cccgcatcac caccgcgtac 1500

gacaaccgcc catcgcacct cccctgaacg cccggcgccg cccttcttgc cgggtcggct 1560

acggctccgc tccccgacac cccccgcgcg ggcgagcgga gccgtagtgc tgtccgcatg 1620

cgtttccgga cgttccgtgg gtcctgtcgt tcc 1653

<210> 7

<211> 35

<212>DNA

<213> 6842up-f

<400> 7

cccaagcttg tggcgtggaa gcagttgagg aaggc 35

<210> 8

<211> 32

<212>DNA

<213> 6842up-r

<400> 8

gctctagaca ccgggagcat ctggttggcg ta 32

<210> 9

<211> 36

<212>DNA

<213> 6842down-f

<400> 9

cggaattcga cgatgtcgag gcttcgtttc tgttcc 36

<210> 10

<211> 33

<212>DNA

<213> 6842down-r

<400> 10

gctctagata ctcggtgtcc aggctggtgg tgg 33

<210> 11

<211> 24

<212>DNA

<213>con6842-f

<400> 11

tgctcgtcga acggatcgca gacc 24

<210> 12

<211> 24

<212>DNA

<213>con6842-r

<400> 12

tggaacgaca ggacccacgg aacg 24

Claims (10)

1. A milbemycin synthesis positive regulatory gene kelR, characterized in that the nucleotide sequence of the kelR gene is as shown in SEQ ID No.1. 2. a kind of milbemycin synthetic positive regulatory protein KelR, is characterized in that, is encoded by the milbemycin synthetic positive regulatory gene kelR of claim 1, and described KelR albumen is made up of 274 amino acids, Its amino acid sequence is shown in SEQ ID No.2. 3. A high-yield milbemycin genetically engineered bacterium, characterized in that, said genetically engineered bacterium overexpresses the milbemycin synthetic positive regulatory gene kelR according to claim 1, and its nucleotide sequence is as SEQ ID No. As shown in 1, the starting strain is one of Streptomyces bingchenggensis, Streptomyces milbemycin, Streptomyces hygroscopicus subsp. 4. A kind of high-yield milbemycin genetically engineered bacteria according to claim 3, characterized in that, the Streptomyces bingchenggensis is Streptomyces bingchenggensis BCWT or Streptomyces bingchenggensis BC-120-4. 5. a preparation method of the high-yield milbemycin genetic engineering bacterium described in claim 3 or 4, is characterized in that, comprises the following steps: 1) Construct the overexpression vector of milbemycin synthesis positive regulation gene kelR, the overexpression vector contains milbemycin synthesis positive regulation gene kelR, its sequence is shown in SEQ ID No.1; and milbemycin synthesis positive Regulatory gene kelR upstream promoter, its sequence is shown in SEQ ID No.3; 2) Transforming the Escherichia coli host cell with the milbemycin synthesis positive regulation gene kelR overexpression vector constructed in step 1), to obtain a transformant containing the milbemycin synthesis positive regulation gene kelR overexpression vector; 3) Intergenus conjugation and transfer of the transformant obtained in step 2) with Streptomyces to obtain a high-yield milbemycin genetically engineered bacterium. 6. the preparation method of the high-yield milbemycin genetically engineered bacterium according to claim 5, is characterized in that, step 1) described milbemycin synthesizes the construction process of the overexpression vector of positive regulatory gene kelR as: Using the Streptomyces glaciferi genome as a template, PCR amplifies the gene fragment containing the complete coding region of the milbemycin synthesis positive regulatory gene kelR and the upstream promoter, and the upstream primer sequence used for the PCR amplification is as SEQ ID No.4 As shown, the sequence of the downstream primer is shown in SEQ ID No.5; the restriction endonuclease XbaI and EcoRI are used to digest, and the obtained digestion product is connected to the pSET152 vector that has been digested with XbaI and EcoRI to obtain the recombinant plasmid pSET152::kelR is the overexpression plasmid of the milbemycin synthesis positive regulatory gene kelR; the Streptomyces bingchenggensis is Streptomyces bingchenggensis BCWT or Streptomyces bingchenggensis BC-120-4. 7. the preparation method of high-yield milbemycin genetically engineered bacteria according to claim 6, is characterized in that, step 3) described streptomyces is Streptomyces bingchenggensis BCWT, Streptomyces bingchenggensisBC-120-4, milbemycin streptomyces , Streptomyces hygroscopicus subsp. aureus, and Streptomyces griseochromogenes. 8. The preparation method of the high-yield milbemycin genetic engineering bacterium according to claim 5, characterized in that, the Escherichia coli host cell described in step 2) is E.coli ET12567\pUZ8002. 9. The application of the milbemycin synthesis positive regulatory gene kelR according to claim 1 in improving the output of milbemycin in the production of milbemycin by microbial fermentation. 10. the application of the high-yield milbemycin genetically engineered bacterium described in claim 3 or 4 in the fermentative production of milbemycin, is characterized in that, the genetically engineered bacterial strain of high-yield milbemycin is cultivated, isolates milbemycin from the culture Bemycin.