CN113355346A - Method for deleting redundant segments on a stable vector, stable vector obtained by said method and use thereof - Google Patents

Abstract

The invention provides a method for deleting redundant segments on a stable vector, the stable vector obtained by the method and application thereof. The method comprises the following steps: 1) introducing a stable vector into a eumycete rokitamuni to obtain a transformant, wherein 1 recombinase-specific recognition nucleotide sequence is respectively arranged at the upstream and downstream of the redundant fragment in the stable vector, and the stable vector comprises a promoter parP in a par region, a plasmid stabilizing factor parA, a parB, a recognition sequence parS, a replication protein repA, and the redundant fragment; 2) and introducing a helper plasmid for expressing the recombinase into the transformant, and deleting the redundant fragment through recombination. The invention can eliminate the potential hidden trouble that the resistance gene is diffused to the nature caused by using the resistance gene in the industrial strain.

Description

Method for deleting redundant segments on a stable vector, stable vector obtained by said method and use thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a method for deleting a stable vector of a redundant fragment on the stable vector and the stable vector obtained by the method.

Background

The fungus Eutropha rolfsii (Ralstonia eutropha, also known as cupriavidius necator) is an important model bacterium for studying PHA (polyhydroxyalkanoate), a completely degradable bio-based material, and has potential as a strain for PHA industrial production. A target gene is delivered to a biological cell (a receptor cell) by genetic engineering means, and a carrier is required to carry the foreign gene into the receptor cell, and the carrier is called a vector (vector), and a plasmid is the most commonly used vector. In the research and development and industrial production of Ralstonia eutropha strains, a plasmid vector is often used to introduce foreign genes to achieve the purpose of optimizing the strains.

The problem of high yields in large scale production and the associated problems that may be encountered in fermentation and subsequent purification are taken into account in the construction of plasmid vectors and in the selection of host bacteria. Plasmid stability is one of the problems frequently encountered in plasmid production, and plasmid instability refers to mutation or loss of recombinant bacteria in a culture process, so that the recombinant bacteria lose the original phenotypic characteristics. Destabilisation of plasmids is the destabilisation caused by the separation of daughter cells after cell division. This is also a significant cause of plasmid loss, as a result of the uneven distribution of plasmid among daughter cells leaving a portion of the cells free of plasmid. In general experiments, selection resistance can be usually applied using antibiotics to maintain plasmid stability, but considering the amount of industrial fermentation, adding antibiotics in actual production greatly increases costs.

The par system is a system for stably maintaining a plasmid, and after the par system is activated, the plasmid is uniformly distributed into daughter cells after replication, so that a strain stably maintaining the plasmid can be obtained even without applying selection resistance by antibiotic resistance (Salje J, Gayathri P,

J.The ParMRC system:molecular mechanisms of plasmid segregation by actin-like filaments[J]nature Reviews Microbiology 2010,8(10): 683-692.). At present, the methodA plasmid vector constructed using the par system, which can be stably maintained without applying antibiotic selection pressure and can be hosted by Ralstonia eutropha, has been developed. The vector used the promoter parP, plasmid stabilizing factors parA28, parB28 and the recognition sequence parS in the par region of the huge plasmid pMOL28 of Cupridopis metalate CH 34. Meanwhile, the vector is provided with an escherichia coli replicon and a kanamycin resistance gene so as to facilitate genetic operation related to vector modification in escherichia coli (see the Chinese patent application publication No. CN 101297036A).

In general, maintaining the presence of plasmid and replication of plasmid depend on the energy produced by the metabolism of the host cell, and the plasmid copy number is too high or large, which can increase the metabolic burden of the host cell and even affect the growth of the host cell (Karim A S, Current K A, Alper H S. characteristics of plasmid and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications [ J ]. Fems Yeast Research, 2013; 13(1): 107-116.). Since the size of the foreign gene is usually not easily regulated, it is necessary to simplify the vector design as much as possible to avoid the replication burden caused by the ineffective gene. In the process of plasmid transformation, the recipient bacteria are often subjected to antibiotic pressure screening by means of resistance genes on the vector to ensure that transformants carrying the plasmid are obtained. However, in the case of a stable plasmid vector, after the plasmid is transferred into a recipient bacterium, the use value of the resistance gene on the vector is lost and the vector becomes an "invalid" fragment because antibiotics are no longer required.

The site-specific recombination system consists of a recognition site/sequence of integrase and integrase which recognizes DNA sequences and mediates DNA recombination. By altering the position and sequence orientation of the recombinase recognition sites, the site-specific recombinase can serve as a tool enzyme for inserting a specific sequence into a DNA strand or deleting or inverting a specific sequence from a DNA strand. The commonly used site-specific recombinases are TP901, PhiC31, P22 and VCre, among others.

The vectors used in the prior art contain a DNA replication initiation region which functions in the genera Ralstonia, Cupriavidus and Waters and a region (par region) which stabilizes the recombinant vector using the promoter parP in the par region of the giant plasmid pMOL28 of the metal-tolerant Cupriavium CH34, the plasmid stabilizing factors parA28, parB28 and the recognition sequence parS. The vector is stable in bacteria using the par system without antibiotic selection pressure. Meanwhile, the carrier is provided with an escherichia coli replicon and a kanamycin resistance gene so as to facilitate genetic operation related to carrier modification in escherichia coli.

The stable vectors of the prior art, although not requiring the addition of antibiotics, still have kanamycin resistance gene present and expressed, which is an extra energy and raw material consumption for DNA replication and protein expression; moreover, the stable vector with the kanamycin resistance gene is applied to industrial production bacteria, and the hidden danger that the kanamycin resistance gene diffuses to the nature exists; in addition, replicons remain on the stable vectors, which are replicable in E.coli, and which are redundant fragments that are no longer functional after the vectors have been transferred into Ralstonia bacteria, and are an excess of energy and material consumed for DNA replication.

Based on the above-described situation, it is necessary to provide a method capable of deleting redundant E.coli replicon and kanamycin resistance gene in the above recombinant vector to provide a stable vector suitable for Ralstonia eutropha.

Disclosure of Invention

It is an object of the present invention to provide a method for deleting redundant fragments on a stable vector.

It is another object of the present invention to provide a stable vector from which redundant fragments are deleted, obtained by the above method.

It is a further object of the present invention to provide a use of the above-mentioned stabilized carrier.

It is still another object of the present invention to provide a recombinant strain of a eubacterium reuteri comprising a stable vector from which redundant fragments have been deleted by the above method.

Still another object of the present invention is to provide the use of the recombinant strain of Eubacterium reuteri for PHA production.

In one aspect, the present invention provides a method for deleting redundant fragments on a stable vector, the method comprising the steps of:

1) introducing a stable vector into the Eubacterium rolfsii to obtain a transformant, wherein the upstream and downstream of the redundant fragment in the stable vector have 1 recombinase-specific recognition nucleotide sequences, and the stable vector comprises a promoter parP in the par region, a plasmid stabilizing factor parA, a parB, a recognition sequence parS, a replication protein repA, and the redundant fragment;

2) and introducing a helper plasmid for expressing the recombinase into the transformant, and deleting the redundant fragment through recombination.

In the method of the present invention, the recombinase may be preferably selected from VCre recombinase, Bxb1 recombinase, phiC31 recombinase, TP901 recombinase, HK022 recombinase, Phi80 recombinase, P21 recombinase, Int3 recombinase, Int4 recombinase, Int5 recombinase, Int9 recombinase, Int11 recombinase, Int12 recombinase, Int13 recombinase, P22 recombinase, lamda recombinase, Cre recombinase, Dre recombinase, Vika recombinase, Flp recombinase, FimE recombinase, HbiF recombinase, and a118 recombinase.

In a specific embodiment, the recombinase is a VCre recombinase and the nucleotide sequence specifically recognized by the recombinase is a VloxP sequence (SEQ ID No.: 20).

In specific embodiments, the amino acid sequence of the VCre recombinase is as set forth in SEQ ID No.: 22, respectively.

In specific embodiments, the nucleotide sequence of the VCre recombinase is as set forth in SEQ ID No.: shown at 21.

In a specific embodiment, the nucleotide sequence of the par region comprises a nucleotide sequence as set forth in SEQ ID No.: 25, or a sequence shown in seq id no.

In a specific embodiment, the replication protein repA is replication protein repA28 comprising a sequence as set forth in SEQ ID No.: 26, or a sequence shown in fig. 26.

In the method of the invention, the optional par sequence may preferably further comprise vector stabilizing regions of RP4, RK 2.

In the present invention, examples of the Eubacterium reuteri are not particularly limited, and may be any of the Eubacterium reuteri used in the art for PHA production. In a specific embodiment, the Eubacterium rolfsii is Ralstonia eutropha H16.

The term "redundant fragment" as used herein refers to a gene fragment and/or nucleotide sequence in which the sequence of the stabilizing vector does not function and/or has no practical significance after introduction into Eubacterium reuteri.

In a specific embodiment, the redundant fragment comprises a resistance gene. Preferably, the resistance gene is an antibiotic resistance gene.

In a specific embodiment, the resistance gene is a kanamycin resistance gene.

In particular embodiments, the redundant fragment further comprises an E.coli replicon.

In specific embodiments, the Ptet inducible promoter may also be replaced by other promoters for expressing VCre, including but not limited to inducible promoters Ptac, pBAD, pra, pTrp, T7 promoter, heat-inducible promoters, etc., as well as constitutive promoters J23119 and other J231xx series, constitutive promoters of Sigma70 factor mediated transcription in prokaryotic cells, etc.

In another aspect, the present invention provides a stable vector suitable for a eubacterium reuteri obtained by the above method.

In still another aspect, the present invention provides the use of the above-described stable vector in genetic engineering.

In a further aspect, the present invention provides the use of a stable vector as described above for use in a eubacterium reuteri.

In still another aspect, the present invention provides a recombinant strain of a eubacterium reuteri comprising a stable vector from which redundant fragments have been deleted by the above-described method.

In still another aspect, the present invention provides the use of the above recombinant strain of Eubacterium reuteri for the production of Polyhydroxyalkanoates (PHA).

Advantageous effects

The invention has the beneficial technical effects that on one hand, redundant DNA replication and protein expression of the escherichia coli replicon and resistance gene when the stable vector is used in the eumycete rolfsii are avoided, so that energy is saved and raw material consumption is reduced; on the other hand, the potential hidden danger that the resistance gene is diffused to the nature because of using the resistance gene in the industrial strain is avoided.

Drawings

FIG. 1 is a schematic diagram of deletion of an E.coli replicon and an antibiotic resistance gene by a vector containing a recombinase VCre gene in a stable vector according to the present invention.

FIG. 2 shows the plasmid retention rate of Ralstonia eutropha H16(pSP-kana-GFP) in example 2, both with and without kanamycin.

FIG. 3 shows the plasmid retention rate of Ralstonia eutropha H16(pBBR1-GFP) in comparative example 2, both with and without kanamycin addition.

FIG. 4 shows the results of PCR before (lanes 1-3)/after (lanes 5-7) deletion of the E.coli p15A replicon and kanamycin resistance gene using the helper plasmid pVCre.

FIG. 5 shows the plasmid retention rates of Ralstonia eutropha H16(pSP-kana-GFP) without deletion of the p15A replicon and kanamycin resistance gene and Ralstonia eutropha H16(pSP-GFP) with deletion of the p15A replicon and kanamycin resistance gene.

Detailed Description

Hereinafter, the present invention will be described in detail by examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The enzymatic reagents used were purchased from ThermoFisher and New England Biolabs (NEB), the kit for plasmid extraction was purchased from Tiangen Biotechnology technology (Beijing) Ltd, the kit for DNA fragment recovery was purchased from omega USA, the corresponding procedures were performed strictly according to the product instructions, and all media were prepared with deionized water if no special instructions were given.

The formula of the culture medium is as follows:

LB culture medium: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042),10g/L NaCl, and the balance water. Adjusting pH to 7.0-7.2, and sterilizing with high pressure steam.

Example 1: preparation of Green Fluorescent Protein (GFP) -integrating plasmid pSP-kana-GFP Using plasmid vector pSP

A fragment containing the p15A replicon and kanamycin resistance gene was amplified using the template of synthetic fragment 01(SEQ ID No.: 15) using primer 1(SEQ ID No.: 1) and primer 2(SEQ ID No.: 2), a fragment containing the par region was amplified using the template of synthetic fragment 02(SEQ ID No.: 16) using primer 3(SEQ ID No.: 3) and primer 4(SEQ ID No.: 4), a fragment containing the par region was amplified using the template of synthetic fragment 03(SEQ ID No.: 17) using primer 7(SEQ ID No.: 5) and primer 8(SEQ ID No.: 6), a fragment containing the replication initiation protein Rep and the restriction endonuclease BsaI site was amplified using the template of synthetic fragment 03(SEQ ID No.: 17), and the three fragments obtained were subjected to a commercial kit (SEQ ID No.: 2)

Master Mix, purchased from New England Biolabs (NEB) company) was ligated by the Gibson Assembly method to obtain a pSP vector with kanamycin resistance, upstream of the p15A replicon and downstream of the kanamycin resistance gene, 1 VCre recombinase (amino acid sequence SEQ ID No.: 22, gene sequence SEQ ID No.: 21) specifically recognized VloxP sequence (SEQ ID No.: 20). The primers used are as in table 1 below:

TABLE 1

Synthesis of fragment 04(SEQ ID No.: 18) including the GFP green fluorescent protein gene was treated with BsaI, and the digested fragment was introduced into the restriction enzyme BsaI site of pSP vector by Golden Gate (Werner S, Engler C, Weber E, et al, fast track assembly of multigene constraction using Golden Gate cloning and the MoClo system [ J ]. Bioengineered plugs, 2012,3(1):38-43.), to obtain pSP-kana-GFP plasmid constructed using pSP vector.

The plasmid pSP-kana-GFP was transferred into E.coli S17-1(ATCC No. 47055, available from American Type Culture Collection), and then transferred into Ralstonia eutropha H16 (China general microbiological Culture Collection center, CGMCC 1.7092) by the ligation transformation method, and LB plate containing 250. mu.g/ml kanamycin and 50. mu.g/ml apramycin at the same time was used to screen positive clones with green fluorescence, thus obtaining a transformant Ralstonia eutropha H16(pSP-kana-GFP) constructed using the pSP vector.

Comparative example 1: preparation of a plasmid pBBR1-GFP incorporating the Green Fluorescent Protein (GFP) Using the vector pBBR1MCS2

PCR was performed using primers 9(SEQ ID No.: 7) and 10(SEQ ID No.: 8) with plasmid pBBR1MCS-2(Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM.,1995.Four new derivatives of the branched-host-range cloning vector pBBR1MCS, and random differential anti-viral-resistance cassettes genes. Gene.166,175-176) as templates to obtain vector fragments carrying replicon and kanamycin resistance genes; amplifying a DNA fragment containing the GFP gene by using a primer 11(SEQ ID No.: 9) and a primer 12(SEQ ID No.: 10) and using the synthesized fragment 04(SEQ ID No.: 18) as a template; the two fragments were ligated by the Gibson Assembly method to obtain a plasmid pBBR1-GFP which integrates Green Fluorescent Protein (GFP) using the plasmid vector pBBR1MCS 2. The primers used are as in table 2 below.

TABLE 2

The plasmid pBBR1-GFP was transferred into Ralstonia eutropha H16 in the same manner as in example 1, whereby Ralstonia eutropha H16(pBBR1-GFP) into which the plasmid pBBR1-GFP was transferred was obtained.

Example 2: plasmid Retention Rate of Ralstonia eutropha H16(pSP-kana-GFP)

The plasmid retention of Ralstonia eutropha H16(pSP-kana-GFP) was identified using flow cytometry. The assay was performed as follows: the transformants were subjected to shake culture (30 ℃ C., 220rpm/min) using LB liquid medium containing 250. mu.g/ml kanamycin and no antibiotic, respectively, and passaged every 24 hours, and the plasmid retention of the transformants after 2, 4, 6, and 8 days of serial passaging was measured.

Firstly, the OD value of the bacterial liquid of the transferred bacterial liquid is adjusted to 0.012 by 10 XPBS (purchased from Beijing Solarbio company, product catalog number P1022), 200 mul of diluted bacterial liquid is absorbed and added into a 96-well plate (purchased from Corning company, product catalog number 351172), the number of cells with green fluorescence in each sample is counted by a flow cytometer (Beckmann Cytoflex), only the transformant which retains pSP-kana-GFP can emit green fluorescence, and the percentage of the transformant which occupies the total number of all cells is the plasmid retention rate. Specific experimental procedures were performed according to the instrument instructions. The results are shown in FIG. 2.

As can be seen from FIG. 2, after continuous transfer of Ralstonia eutropha H16(pSP-kana-GFP) without addition of kanamycin resistance, no significant loss of plasmid occurred, and the plasmid retention remained above 90% after 8 days of transfer, which is not significantly different from transfer with addition of kanamycin. From the above results, it is considered that the pSP vector can be stably maintained without antibiotic application.

Comparative example 2: plasmid Retention of Ralstonia eutropha H16(pBBR1-GFP)

The plasmid retention was determined in the same manner as in example 2, except that Ralstonia eutropha H16(pBBR1-GFP) was used in place of Ralstonia eutropha H16(pSP-kana-GFP) in example 1. The results are shown in FIG. 3.

As can be seen from FIG. 3, the plasmid was significantly lost after continuous transfer of Ralstonia eutropha H16(pBBR1-GFP) without addition of kanamycin resistance, and only less than 10% of the transformants retained the pBBR1-GFP plasmid after 8 days of transfer, which is significantly different from transfer with addition of kanamycin resistance. From the above results, it is considered that the pBBR1MCS2 vector could not be stably maintained without applying antibiotic selection pressure.

Example 3: deletion of the kanamycin resistance Gene on the pSP-kana-GFP plasmid Using the helper plasmid

Carrying out PCR amplification by using a primer 13(SEQ ID No.: 11) and a primer 14(SEQ ID No.: 12) by using a plasmid pBBR1MCS2 as a template to obtain a vector fragment, wherein the fragment carries a replicon and a kanamycin resistance gene; amplifying a DNA fragment containing a Ptet inducible promoter, a VCre gene (SEQ ID No.: 21) and a spectinomycin resistance gene by using a primer 15(SEQ ID No.: 13) and a primer 16(SEQ ID No.: 14) and a synthetic fragment 05(SEQ ID No.: 19) as a template; the two fragments were ligated by the Gibson Assembly method to obtain the helper plasmid pVCre. The primers used are as in table 3 below.

TABLE 3

The helper plasmid pVCre was transferred into E.coli S17-1, the transformant obtained in example 1 was transferred by the conjugal transformation method, 3 clones with green fluorescence were randomly picked up using LB plates containing both 500. mu.g/ml spectinomycin and 50. mu.g/ml apramycin, PCR verification was performed using primer 17(SEQ ID No.: 23) and primer 18(SEQ ID No.: 24) to obtain the expected band of 1104bp, and the transformant Ralstonia eutropha H16(pSP-kana-GFP) was amplified using the same primers as a control to obtain the expected band of 3074bp (FIG. 4), demonstrating that the helper plasmid pVCre containing the introduced gene of VCre recombinase deletes the p15A replicon and kanamycin resistance gene whose fragment lengths are 1970 bp. Finally, the positive clones were cultured on a non-resistant plate to lose the pVCre plasmid, resulting in the pSP vector transformant Ralstonia eutropha H16(pSP-GFP) without the p15A replicon and kanamycin resistance gene.

Example 4: plasmid Retention of Ralstonia eutropha H16(pSP-GFP)

Plasmid retention was determined in the same manner as in example 2 using Ralstonia eutropha H16(pSP-GFP) under non-resistant conditions, while using Ralstonia eutropha H16(pSP-kana-GFP) as a control. The results are shown in FIG. 5.

As can be seen from FIG. 5, there was no significant loss of plasmid after continuous transfer of Ralstonia eutropha H16(pSP-GFP) without addition of kanamycin resistance, which was not significantly different from Ralstonia eutropha H16 (pSP-kana-GFP). From the above results, it is considered that pSP-kana-GFP could be stably maintained even after deletion of the kanamycin resistance gene and the p15A replicon.

In summary, the vector constructed by the present application, in which the p15A replicon and the kanamycin resistance gene were deleted, had no significant difference in stability retention rate with respect to the vector in which the above genes were not deleted, and in addition, the stable vector constructed by the present application eliminated the risk of the kanamycin resistance gene diffusing into the natural world.

Sequence listing

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aagtaccggg ggaacagctc gatctctcgg gtacaacagg cacaaagtta acttgcgtat 720

acgtctaagg gccgctaacc ttcacggcaa cgcaaccgcg gacgtcattt ttgccgaaaa 780

cggttgcacg atccaccggc ggttccggtg aacagcttaa aggtttttga accgaatgga 840

tatcaagcta gcccacccca gcaagacgac gcctgaggat ttgaagcagt tggcaaatct 900

ctccgcggtg atgctgcaga aaattcggga tgagatgctg gagccatttc ctcggaagga 960

agccccgctg atcccgtctg gccgcctaca agaattgtgt ggcatcgaca aaacgcggat 1020

gaaccggtcc ctcaaaaagg gggatctccc tcagggccag caatcgcgac ccggtgcagt 1080

gcgctatttc agcctcagcg aggcaatgca atggatccga gcggaactta agcctgtccc 1140

gcgaagggga ccaggtaaag tcattgcagt tgcgaacttc aagggcggtg tcacgaagac 1200

cactatgtcc accctcctct gccagggctt gagtctgcgg cgaggtcgga aggtgtgcca 1260

cgttgatctg gatccgcagg gaagcgcaac cacgctgtat ggcatcaatc cacatgccga 1320

ggtgtcgtcc gaaaacacca ttatgccgct catcgaggcg tatttggcgg gcgagtcctt 1380

cgatatgcga gggcttcctc aggagactta ctggcctaac ctggatttga ttccttcgtc 1440

tactgagctt ttcaacgcgg agtttatgct tccggctcgg gcgacggcag aggaaggcca 1500

tattccgttc gagcgcgtgt taagtaacgg cctcgattcg ttgaaagacg aatatgacta 1560

catcatcctc gacacggctc ctaccctcag ctacctgacc atcaacgcga ttttcgctgc 1620

cgatggcgtc atcgtaccgg tggtcccgga caccttggct ttcgcgtcta tggtccagtt 1680

ctggcaactc ttctcggacc tagtaacagg catggaagag cagagcgagg gatctaaaaa 1740

ggagttcgac tttctcgatg ttctcatgac acgcatggag aaaaagaacg ctcctcgcct 1800

ggtggcagac tggattcgcg gcgtctatgg gtcgcgcgtg ctgccgattg agatccctga 1860

gacggacctc gcccgtaaca gcagcattca atttcgcacg gtctatgacc tctcctctag 1920

cgaggcgaac accgagacga tgcgacgcat tcgccaaccc tgcgatgagt ttgtcgacta 1980

tgtggacgac aaggtcagcg cgctttggca aggaattgaa gaatgagttt gagagaaaag 2040

cttgccgcaa aggctgggaa catcaaggtc acggcggaag acttggagaa agccgctgcg 2100

cgcggtccgc aagcgccgcg aactgcgccc ggtcagttaa tgcatatgca agggaaggtt 2160

gagcgacagg ctaacgagat cgcgcaacta agagcagaac ttgagtcggc ccgcgtcagc 2220

ggcggcgcag tggatgtgcc tatcgaccaa ctgcatgagg tcccaggccg cagacgcttc 2280

atgcctcccg agaagtatgt cgaattgagg gaaaacctca ggcacaacaa gctcgttcat 2340

cctgtgattg tatgccctcg gcctgcggga ggcttcgaga ttgtctccgg gcatcaccgg 2400

acagacgcgt accgcgagct tgggcgcgat cacatacgct gcgtgctcgg cgaacttagt 2460

tcagacgagg ctgacacggg cgcgttctac gcgaacctta tgc 2503

<210> 17

<211> 2381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of fragment 03

<400> 17

cgttctacgc gaaccttatg cagtcagatt taacggattt cgagaagttt cggaagttcg 60

acgaactgct gcttcgcagc ccagacaaga ctcaagccgc aatagctgaa caggctggtg 120

tacctgtctc gactctctca gagattttgt cgttccggaa cttgcctccc gaggtcctaa 180

gccttctcga tagccgccca gacctgctcg ggtcgaatgc tggcgccgag ttggcaaggg 240

cgaccaaaga cggtcgcggg gatcgggtcg tcgaagcggt taagttgttg gccgagaaga 300

agatcgatca acagcaggcc gtacggatga ctaaggccga gcaggttaag accaggcctg 360

ccgcatctac cggcttcaaa atcaaggcgg gaaaggcgac ttggtgcgat gttcgtatcg 420

caaagaaagt catgcgcatt gagttccgca gcgaggaaga agcggaagcg gcccaatcgg 480

ccattcgcga acatctggaa gggttagcta aagctgcgtc ggaagacgca aaaagctaag 540

tgcttgtttt ttaaggactt cgtactacga atcgaggttt taagccatgt ctagactgta 600

atcctacaaa aacaaaagcc cacggcggca accgtgggct tttgagaact tcaagctgac 660

cagtttcccg gccgctacac accgaagcca ctcgacatgg attcgttagt cggagtgtag 720

cggaacgcga acctgagtca agcgacttca accatttttt acgaatggga aggtcatatg 780

actttcgcgc aacgctccgt tgctggcgag gatgttgctc gcccacaaaa acaccttaac 840

cagacagacg ctctgattgc tccggcgccc aagcgcctca aacgcaagac tatcgaagca 900

gtcgagcgcg caactcgaat cgtcgggatc gggcgtagcg cccgatcagc tcttgccgcc 960

ctcgcccgca cggcgaataa cgatgacccc accggtaaga tctttaagca ccgggaaacg 1020

ctttgtgccg aaaccggaat gtcgccggct acttggtacc gtgctcaacg agaactgctc 1080

gacttgggcc taattaccgt cgacgttcaa gttcggaagc gatttggccg attcgcagga 1140

gcctacattt acctgacgga aaaagcgacg gagatgctcg gcttaagctc gcgaaaagaa 1200

gaagaaacca cgggtacggg cgaggacgac acagcgcagc tcggcgagcc ggccgttcca 1260

ccaccctctt ctatggcgca accgtctctc aaaacgagag tcctgtttac agaagatcgt 1320

gtcccatact cctttcaaaa aagacagcag gatcggctcc cccaggacct gacacgtctg 1380

cgcggcctgg gtcttgatgt aaatttaatt ttttggttga tgcgaaaggc taaagagcaa 1440

ggccactttc tctcagatgt cgtaagcgcg acatgggaga gtcttgcgaa agcacgcgtg 1500

ccaaaagcgt atctgcttgc cctactcacc gcccgcaccg atttcagtgc tgtctgcaaa 1560

gcaaaggcac tcaaagaaga caaagcccga atccaagtgc aggaccgcga tttcgtgcgt 1620

tcgatactcg caggggcagc gcggcagtgt ttcgtggacg aaaaaggcaa ccatttcgaa 1680

gtcgaaagcg acggaagctc agtgcttgtc accgaggtcc aaagtgcggt cacttcccgc 1740

ttggtaggaa cttccctcgc cgaatttgca aggcgactac acgctggtgc gtaccagaaa 1800

gctgaggtct acgctgctcc ccagagagca agcggccggc tcgagaaacg gggaaaggag 1860

gcggcttcga cgttatcggc gttgcgagcg atgctgcgcg accgcaggtc agccaacgcg 1920

gcaaacacga cgaacaatgc tcatgccatg gcctagggtg tgttttgcgc tgaaagtcta 1980

gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa 2040

ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctttaattaa agcggataac 2100

aatttcacac aggagagtga agagcttttg ctcttcatcc aagcgagacc aaaaggtctc 2160

gccgcgaccc attaccgcct ttgagtgagc gtcgtgactg ggaaaaccct ggcgactagt 2220

cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga 2280

acgctcggtt gccgccgggc gttttttatt ggtgagaatc cagtcaattt ccgagaatga 2340

cagttctcag aaattgaacg tctcattttc gccagatatc g 2381

<210> 18

<211> 1549

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of fragment 04

<400> 18

agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca 60

aataattttg tttaagagcg gcgagacctt tcacacagga atgcctccac accgctcgtc 120

acatcctgtt gcgttcactg gaatcccagt atagactttg acctgcgagc aagctgtcac 180

cggatgtgct ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt 240

gtttaagagt tactagagaa agaggagaaa tactagatgc gtaaaggcga agagctgttc 300

actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc 360

gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt 420

actactggta aactgccggt accttggccg actctggtaa cgacgctgac ttatggtgtt 480

cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg 540

ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg 600

cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt 660

gactttaaag aagacggcaa tatcctgggc cataagctgg aatacaattt taacagccac 720

aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc 780

cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc 840

ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct 900

aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc 960

atcacgcatg gtatggatga actgtacaaa taatcgtcac tccaccggtg cttaataacc 1020

aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 1080

ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc 1140

gtttatatac tagagctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg 1200

ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 1260

tgaaaggagg aactatatcc ggattactag aggtcatgct tgccatctgt tttcttgcaa 1320

gatgctcact caaaggcggt aatgggtcga tgaagagcaa aagctcttca ctcgtcgtga 1380

ctgggaaaac cctggcggtc tcgcttggac tcctgttgat agatccagta atgacctcag 1440

aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga 1500

atccaggggt ccccaataat tacgatttaa attggcgaaa atgagacgt 1549

<210> 19

<211> 2185

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of fragment 05

<400> 19

cgtctcattt tcgccagata tcgacgtctt aagacccact ttcacattta agttgttttt 60

ctaatccgca tatgatcaat tcaaggccga ataagaaggc tggctctgca ccttggtgat 120

caaataattc gatagcttgt cgtaataatg gcggcatact atcagtagta ggtgtttccc 180

tttcttcttt agcgacttga tgctcttgat cttccaatac gcaacctaaa gtaaaatgcc 240

ccacagcgct gagtgcatat aatgcattct ctagtgaaaa accttgttgg cataaaaagg 300

ctaattgatt ttcgagagtt tcatactgtt tttctgtagg ccgtgtacct aaatgtactt 360

ttgctccatc gcgatgactt agtaaagcac atctaaaact tttagcgtta ttacgtaaaa 420

aatcttgcca gctttcccct tctaaagggc aaaagtgagt atggtgccta tctaacatct 480

caatggctaa ggcgtcgagc aaagcccgct tattttttac atgccaatac aatgtaggct 540

gctctacacc tagcttctgg gcgagtttac gggttgttaa accttcgatt ccgacctcat 600

taagcagctc taatgcgctg ttaatcactt tacttttatc taatctagac atcattaatt 660

cctaattttt gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat 720

agagaaaaga attcaagctg tcaccggatg tgctttccgg tctgatgagt ccgtgaggac 780

gaaacagcct ctacaaataa ttttgtttaa tactagagaa agaggagaaa tactagatga 840

tcgagaacca gctgagcctg ctgggtgatt tcagcggcgt gcgtccggac gatgttaaga 900

ccgcgatcca ggcggcgcaa aagaaaggta ttaacgttgc ggagaacgaa caattcaaag 960

cggcgtttga gcacctgctg aacgagttca agaaacgtga ggaacgttac agcccgaaca 1020

ccctgcgtcg tctggaaagc gcgtggacct gctttgtgga ttggtgcctg gcgaaccatc 1080

gtcacagcct gccggcgacc ccggacaccg ttgaggcgtt ctttatcgaa cgtgcggagg 1140

aactgcaccg taacaccctg agcgtgtacc gttgggcgat tagccgtgtt catcgtgttg 1200

cgggttgccc ggacccgtgc ctggatatct atgtggagga tcgtctgaag gcgattgcgc 1260

gtaagaaagt gcgtgagggc gaagcggtta aacaggcgag cccgtttaac gaacaacacc 1320

tgctgaagct gaccagcctg tggtaccgta gcgacaaact gctgctgcgt cgtaacctgg 1380

cgctgctggc ggtggcgtat gagagcatgc tgcgtgcgag cgaactggcg aacatccgtg 1440

ttagcgacat ggagctggcg ggtgatggca ccgcgattct gaccatcccg attaccaaga 1500

ccaaccacag cggcgagccg gacacctgca ttctgagcca ggatgtggtt agcctgctga 1560

tggactacac cgaagcgggc aagctggaca tgagcagcga tggtttcctg tttgtgggcg 1620

ttagcaaaca caacacctgc atcaagccga agaaagataa acagaccggt gaagttctgc 1680

acaagccgat taccaccaaa accgtggagg gcgttttcta tagcgcgtgg gaaaccctgg 1740

atctgggtcg tcaaggcgtg aagccgttta ccgcgcacag cgcgcgtgtt ggtgcggcgc 1800

aggacctgct gaagaaaggc tacaacaccc tgcaaatcca gcaaagcggt cgttggagca 1860

gcggcgcgat ggttgcgcgt tatggtcgtg cgatcctggc gcgtgacggc gcgatggcgc 1920

acagccgtgt gaaaacccgt agcgcgccga tgcaatgggg caaggacgag aaagattaat 1980

gataagtagc ggagtgtata ctggcttacg tcgtgactgg gaaaaccctg gcgactagtc 2040

ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa 2100

cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aggggtcccc aataattacg 2160

atttaaattg gcgaaaatga gacgt 2185

<210> 20

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> VloxP sequence

<400> 20

tcaatttccg agaatgacag ttctcagaaa ttga 34

<210> 21

<211> 1143

<212> DNA

<213> Artificial sequence

<220>

<223> gene sequence of VCre recombinase

<400> 21

atgatcgaga accagctgag cctgctgggt gatttcagcg gcgtgcgtcc ggacgatgtt 60

aagaccgcga tccaggcggc gcaaaagaaa ggtattaacg ttgcggagaa cgaacaattc 120

aaagcggcgt ttgagcacct gctgaacgag ttcaagaaac gtgaggaacg ttacagcccg 180

aacaccctgc gtcgtctgga aagcgcgtgg acctgctttg tggattggtg cctggcgaac 240

catcgtcaca gcctgccggc gaccccggac accgttgagg cgttctttat cgaacgtgcg 300

gaggaactgc accgtaacac cctgagcgtg taccgttggg cgattagccg tgttcatcgt 360

gttgcgggtt gcccggaccc gtgcctggat atctatgtgg aggatcgtct gaaggcgatt 420

gcgcgtaaga aagtgcgtga gggcgaagcg gttaaacagg cgagcccgtt taacgaacaa 480

cacctgctga agctgaccag cctgtggtac cgtagcgaca aactgctgct gcgtcgtaac 540

ctggcgctgc tggcggtggc gtatgagagc atgctgcgtg cgagcgaact ggcgaacatc 600

cgtgttagcg acatggagct ggcgggtgat ggcaccgcga ttctgaccat cccgattacc 660

aagaccaacc acagcggcga gccggacacc tgcattctga gccaggatgt ggttagcctg 720

ctgatggact acaccgaagc gggcaagctg gacatgagca gcgatggttt cctgtttgtg 780

ggcgttagca aacacaacac ctgcatcaag ccgaagaaag ataaacagac cggtgaagtt 840

ctgcacaagc cgattaccac caaaaccgtg gagggcgttt tctatagcgc gtgggaaacc 900

ctggatctgg gtcgtcaagg cgtgaagccg tttaccgcgc acagcgcgcg tgttggtgcg 960

gcgcaggacc tgctgaagaa aggctacaac accctgcaaa tccagcaaag cggtcgttgg 1020

agcagcggcg cgatggttgc gcgttatggt cgtgcgatcc tggcgcgtga cggcgcgatg 1080

gcgcacagcc gtgtgaaaac ccgtagcgcg ccgatgcaat ggggcaagga cgagaaagat 1140

taa 1143

<210> 22

<211> 380

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of VCre recombinase

<400> 22

Met Ile Glu Asn Gln Leu Ser Leu Leu Gly Asp Phe Ser Gly Val Arg

1 5 10 15

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

20 25 30

Asn Val Ala Glu Asn Glu Gln Phe Lys Ala Ala Phe Glu His Leu Leu

35 40 45

Asn Glu Phe Lys Lys Arg Glu Glu Arg Tyr Ser Pro Asn Thr Leu Arg

50 55 60

Arg Leu Glu Ser Ala Trp Thr Cys Phe Val Asp Trp Cys Leu Ala Asn

65 70 75 80

His Arg His Ser Leu Pro Ala Thr Pro Asp Thr Val Glu Ala Phe Phe

85 90 95

Ile Glu Arg Ala Glu Glu Leu His Arg Asn Thr Leu Ser Val Tyr Arg

100 105 110

Trp Ala Ile Ser Arg Val His Arg Val Ala Gly Cys Pro Asp Pro Cys

115 120 125

Leu Asp Ile Tyr Val Glu Asp Arg Leu Lys Ala Ile Ala Arg Lys Lys

130 135 140

Val Arg Glu Gly Glu Ala Val Lys Gln Ala Ser Pro Phe Asn Glu Gln

145 150 155 160

His Leu Leu Lys Leu Thr Ser Leu Trp Tyr Arg Ser Asp Lys Leu Leu

165 170 175

Leu Arg Arg Asn Leu Ala Leu Leu Ala Val Ala Tyr Glu Ser Met Leu

180 185 190

Arg Ala Ser Glu Leu Ala Asn Ile Arg Val Ser Asp Met Glu Leu Ala

195 200 205

Gly Asp Gly Thr Ala Ile Leu Thr Ile Pro Ile Thr Lys Thr Asn His

210 215 220

Ser Gly Glu Pro Asp Thr Cys Ile Leu Ser Gln Asp Val Val Ser Leu

225 230 235 240

Leu Met Asp Tyr Thr Glu Ala Gly Lys Leu Asp Met Ser Ser Asp Gly

245 250 255

Phe Leu Phe Val Gly Val Ser Lys His Asn Thr Cys Ile Lys Pro Lys

260 265 270

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

275 280 285

Thr Val Glu Gly Val Phe Tyr Ser Ala Trp Glu Thr Leu Asp Leu Gly

290 295 300

Arg Gln Gly Val Lys Pro Phe Thr Ala His Ser Ala Arg Val Gly Ala

305 310 315 320

Ala Gln Asp Leu Leu Lys Lys Gly Tyr Asn Thr Leu Gln Ile Gln Gln

325 330 335

Ser Gly Arg Trp Ser Ser Gly Ala Met Val Ala Arg Tyr Gly Arg Ala

340 345 350

Ile Leu Ala Arg Asp Gly Ala Met Ala His Ser Arg Val Lys Thr Arg

355 360 365

Ser Ala Pro Met Gln Trp Gly Lys Asp Glu Lys Asp

370 375 380

<210> 23

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> primer 17

<400> 23

aaggcgagga attctacgaa agtgccatat aacgcac 37

<210> 24

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer 18

<400> 24

gcgtcagttt accattagtt gcgtcacctt cacc 34

<210> 25

<211> 2298

<212> DNA

<213> Artificial sequence

<220>

<223> par region

<400> 25

atggatatca agctagccca ccccagcaag acgacgcctg aggatttgaa gcagttggca 60

aatctctccg cggtgatgct gcagaaaatt cgggatgaga tgctggagcc atttcctcgg 120

aaggaagccc cgctgatccc gtctggccgc ctacaagaat tgtgtggcat cgacaaaacg 180

cggatgaacc ggtccctcaa aaagggggat ctccctcagg gccagcaatc gcgacccggt 240

gcagtgcgct atttcagcct cagcgaggca atgcaatgga tccgagcgga acttaagcct 300

gtcccgcgaa ggggaccagg taaagtcatt gcagttgcga acttcaaggg cggtgtcacg 360

aagaccacta tgtccaccct cctctgccag ggcttgagtc tgcggcgagg tcggaaggtg 420

tgccacgttg atctggatcc gcagggaagc gcaaccacgc tgtatggcat caatccacat 480

gccgaggtgt cgtccgaaaa caccattatg ccgctcatcg aggcgtattt ggcgggcgag 540

tccttcgata tgcgagggct tcctcaggag acttactggc ctaacctgga tttgattcct 600

tcgtctactg agcttttcaa cgcggagttt atgcttccgg ctcgggcgac ggcagaggaa 660

ggccatattc cgttcgagcg cgtgttaagt aacggcctcg attcgttgaa agacgaatat 720

gactacatca tcctcgacac ggctcctacc ctcagctacc tgaccatcaa cgcgattttc 780

gctgccgatg gcgtcatcgt accggtggtc ccggacacct tggctttcgc gtctatggtc 840

cagttctggc aactcttctc ggacctagta acaggcatgg aagagcagag cgagggatct 900

aaaaaggagt tcgactttct cgatgttctc atgacacgca tggagaaaaa gaacgctcct 960

cgcctggtgg cagactggat tcgcggcgtc tatgggtcgc gcgtgctgcc gattgagatc 1020

cctgagacgg acctcgcccg taacagcagc attcaatttc gcacggtcta tgacctctcc 1080

tctagcgagg cgaacaccga gacgatgcga cgcattcgcc aaccctgcga tgagtttgtc 1140

gactatgtgg acgacaaggt cagcgcgctt tggcaaggaa ttgaagaatg agtttgagag 1200

aaaagcttgc cgcaaaggct gggaacatca aggtcacggc ggaagacttg gagaaagccg 1260

ctgcgcgcgg tccgcaagcg ccgcgaactg cgcccggtca gttaatgcat atgcaaggga 1320

aggttgagcg acaggctaac gagatcgcgc aactaagagc agaacttgag tcggcccgcg 1380

tcagcggcgg cgcagtggat gtgcctatcg accaactgca tgaggtccca ggccgcagac 1440

gcttcatgcc tcccgagaag tatgtcgaat tgagggaaaa cctcaggcac aacaagctcg 1500

ttcatcctgt gattgtatgc cctcggcctg cgggaggctt cgagattgtc tccgggcatc 1560

accggacaga cgcgtaccgc gagcttgggc gcgatcacat acgctgcgtg ctcggcgaac 1620

ttagttcaga cgaggctgac acgggcgcgt tctacgcgaa ccttatgcag tcagatttaa 1680

cggatttcga gaagtttcgg aagttcgacg aactgctgct tcgcagccca gacaagactc 1740

aagccgcaat agctgaacag gctggtgtac ctgtctcgac tctctcagag attttgtcgt 1800

tccggaactt gcctcccgag gtcctaagcc ttctcgatag ccgcccagac ctgctcgggt 1860

cgaatgctgg cgccgagttg gcaagggcga ccaaagacgg tcgcggggat cgggtcgtcg 1920

aagcggttaa gttgttggcc gagaagaaga tcgatcaaca gcaggccgta cggatgacta 1980

aggccgagca ggttaagacc aggcctgccg catctaccgg cttcaaaatc aaggcgggaa 2040

aggcgacttg gtgcgatgtt cgtatcgcaa agaaagtcat gcgcattgag ttccgcagcg 2100

aggaagaagc ggaagcggcc caatcggcca ttcgcgaaca tctggaaggg ttagctaaag 2160

ctgcgtcgga agacgcaaaa agctaagtgc ttgtttttta aggacttcgt actacgaatc 2220

gaggttttaa gccatgtcta gactgtaatc ctacaaaaac aaaagcccac ggcggcaacc 2280

gtgggctttt gagaactt 2298

<210> 26

<211> 92

<212> DNA

<213> Artificial sequence

<220>

<223> replication protein repA28

<400> 26

ggtcttgatg taaatttaat tttttggttg atgcgaaagg ctaaagagca aggccacttt 60

ctctcagatg tcgtaagcgc gacatgggag ag 92

Claims (10)

1. A method of deleting redundant fragments on a stable vector, the method comprising the steps of:

1) introducing a stable vector into a eubacterium roche to obtain a transformant, wherein the stable vector has 1 recombinase-specific recognition nucleotide sequence upstream and downstream of the redundant fragment, and the stable vector comprises a promoter parP in a par region, a plasmid stabilizing factor parA, a parB, a recognition sequence parS, a replication protein repA, and the redundant fragment, preferably, the nucleotide sequence of the par region comprises a nucleotide sequence as set forth in SEQ ID No.: 25;

2) and introducing a helper plasmid for expressing the recombinase into the transformant, and deleting the redundant fragment through recombination.

2. The method of claim 1, wherein the recombinase is selected from the group consisting of VCre recombinase, Bxb1 recombinase, phiC31 recombinase, TP901 recombinase, HK022 recombinase, Phi80 recombinase, P21 recombinase, Int3 recombinase, Int4 recombinase, Int5 recombinase, Int9 recombinase, Int11 recombinase, Int12 recombinase, Int13 recombinase, P22 recombinase, lamda recombinase, Cre recombinase, Dre recombinase, Vika recombinase, Flp recombinase, FimE recombinase, HbiF recombinase, and a118 recombinase.

3. The method of claim 1, wherein the recombinase specifically recognizes the nucleotide sequence VloxP sequence and the recombinase is VCre recombinase.

4. The method of claim 1, wherein the Eubacterium rolfsii is Ralstonia eutropha H16.

5. The method according to claim 1, wherein the redundant fragment comprises a resistance gene, preferably the resistance gene is an antibiotic resistance gene, more preferably the resistance gene is a kanamycin resistance gene.

6. The method of claim 5, wherein the redundant fragment further comprises an E.coli replicon.

7. A stable vector obtainable by the method of any one of claims 1 to 6.

8. Use of the stable vector of claim 7 in genetic engineering.

9. Use of the stable vector of claim 7 for a eubacterium reuteri.

10. A recombinant strain of Eubacterium reuteri comprising the stable vector of claim 7.

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