CN107699550A - One group of Burkholderia homologous recombination enzyme and its expression vector and application - Google Patents

Abstract

The invention discloses one group of homologous recombination enzyme that can efficiently mediate Burkholderia In vivo recombination to occur, including albumen Red α 7029 with an exonuclease function and albumen Red β 7029 with single-stranded protein function of annealing.The invention also discloses a kind of-Km of expression vector pBBR1 Rha BA 7029 containing the homologous recombination enzyme, and the homologous recombination enzyme is with being incorporated in the application of application and the homologous recombination enzyme in compound glidopeptin or rhizopeptin is obtained improved in Burkholderia In vivo recombination efficiency from E.coli double-stranded DNA Exonucleolytic enzyme level albumen Red γ.The present invention can be used for iteration and delete multiple genes so as to realize that the genome of Burkholderia is simplified, or insertions function promoter carrys out activation unknown synthetic gene cluster in situ etc., and the bioprospecting and functional genomics research to Burkholderia are significant.

Description

One group of Burkholderia homologous recombination enzyme and its expression vector and application

Technical field

The present invention relates to one group of Gram-negative bacteria homologous recombination enzyme and its expression vector and application, more particularly to one group primary Kirschner bacterium homologous recombination enzyme and its expression vector, recombination system and its structure and application in Burkholderia, belong to microorganism base Because of engineering field.

Background technology

Bacterium is the important sources of medicine and agricultural chemicals.In recent years, the gene order-checking engineerings of bacterium have disclosed largely Natural products route of synthesis is not exploited, and these biosynthesis pathways do not excavated can provide new sample for drug screening Storehouse.Excavation to these compounds has two kinds of processing methods, original position activation and heterogenous expression.It is enterprising in natural products genome The deletion and insertion of the simple and direct genetic manipulation mode such as gene of row are the main policies of activation in situ, but most a couple of days so far The producer of right product still lacks simple and direct efficient genetic tool to meet the genome times afterwards comprehensively that genome is excavated.

Homologous recombination technique is gene targeting and the important technology of genome project, Red/ET recombinant techniques be it is a kind of first Used in Escherichia coli, can realize that the heredity to genetic modification is grasped by short homology arm (~50bp) in vivo Make means.Red/ET recombinant techniques are with from red α/red β of bacteriophage lambda Red operators either Rac prophges It is operated based on recE/recT expression^(1-3).The RecE and RecT of total length are sent out between can mediating two linear DNAs Raw homologous recombination (LLHR), thus can be used to clone large fragment DNA to expression from digestion genomic DNA after purification On carrier⁽⁴⁾.Red α and Red β can mediate the generation of a wire and the homologous recombination of a ring-shaped DNA molecule (LCHR), It thus can be used for completing the transformation to cyclic DNA.The Red α beta systems of Escherichia coli can be in some Gram-negative bacterias Middle carry out genetic modification, such as salmonella (Salmonella enterica)⁽⁵⁾, soil Agrobacterium (Agrobacterium tumefaciens)⁽⁶⁾With the Burkholderia (Burkholderia) of Natural Transformation^(7,8), but for many affiliations farther out The function of the mushroom system is then restricted, such as in Yersinia (Yersinia pseudotuberculosi s)⁽⁹⁾, branch tubercle bacillus (Mycobacterium tuberculosi)^(10,11), pseudomonas syringae (Pseudomonas syringae)⁽¹²⁾, Lactococcus lactis (Lactococcus lactis)⁽¹³⁾, C.perfringens (Clostridium perfringens)⁽¹⁴⁾, luminous bacillus (Photorhabdus luminescens)⁽¹⁵⁾And the primary kirschner of non-natural conversion Bacterium⁽⁸⁾In from Escherichia coli Red/ET restructuring enzyme system do not work then.

Burkholderia mesh is a mesh of Proteobacteria, including with a pathogenic category, such as glander-like disease Burkholderia With onion Burkholderia；With an environmentally friendly category, such as plant growth-promoting bacteria P.phytofirmans PsJN and P.rhizoxinica.The DSM 7029 (Polyangium brachysporum DSM 7029) not being classified in addition is fallen within Burkholderia mesh, it is used for the heterologous host as slime bacteria secondary metabolite Epothilones in recent years, and shows conduct The potentiality of the general heterologous host of slime bacteria secondary metabolite expression.Analyzed by antiSMASH, pathogenic primary kirschner Bacterium, environmentally friendly Burkholderia and Burkholderia DSM 7029 include substantial amounts of unknown natural products biosynthesis base Because of cluster, predict that these gene clusters can encode different types of compound, such as Non-ribosomal peptide, polyketide, iron carry Body and terpene.According to statistics, polyketide synthase approach and Non-ribosomal peptide approach are only less than actinomyces in Burkholderia, compare gemma Bacillus, cyanobacteria, slime bacteria and fungi are more, therefore a large amount of new natural products occur in Burkholderia.Although primary kirschner Many natural products have been observed that in bacterium, but many gene clusters be still silence or product do not know, original position activation and inactivation These gene clusters, are to determine and obtain the effective ways of these unknown gene cluster products, and original position activation and inactivated gene then need The genetic manipulation instrument of genetic manipulation can be completed in vivo by establishing.Red/ET restructuring enzyme systems from Escherichia coli can be with Thailand's Burkholderia (Burkholderia thailandensis) and glander-like disease Burkholderia (B.pseudomallei) are entered The knockout of row genome sequence and subclone etc. operate, but it can not but work in the Burkholderia of non-natural conversion, base Develop in this and establish effective Burkholderia genetic manipulation instrument and be particularly important.It is relevant efficiently to mediate through retrieval The homologous recombination enzyme and its expression vector that Burkholderia In vivo recombination occurs, recombination system and its structure in Burkholderia with Using having not been reported.

Bibliography：

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The content of the invention

In view of the shortcomings of the prior art, it is an object of the invention to provide one kind can efficiently mediate Burkholderia In vivo recombination The homologous recombination enzyme and its expression vector of generation, recombination system and its structure and application in Burkholderia.

One group of the present invention can efficiently mediate the homologous recombination enzyme that Burkholderia In vivo recombination occurs, including a tool There is the albumen of exonuclease function (a kind of to be based on (the Burkholderiales strain DSM of Burkholderia DSM 7029 Exonuclease in 7029, Polyangium brachysporum DSM 7029 (=K481-B101=ATCC53,080)) Wp_047194557.1, Red α 7029 and an albumen wp_053013464.1 with single-stranded annealing protein function are named as, It is named as Red β 7029；Wherein：The mrna length of the Red α 7029 is 660 base-pairs, its nucleotide sequence such as SEQ ID Shown in No.1,219 amino acid encodings of the gene code, shown in its amino acid sequence SEQ ID No.5；The Red β 7029 Mrna length be 873 base-pairs, its nucleotide sequence is as shown in SEQ ID No.2,291 amino acid of the gene code Coding, shown in its amino acid sequence SEQ ID No.6.

Above-mentioned sequence SEQ ID No.1 and SEQ ID No.2 nucleotide sequence can be using DSM7029 genomic DNAs mould Plate is obtained by PCR (PCR).DSM7029 is transferred to by electricity after plasmid enzyme restriction checking is correct.

The invention discloses a kind of expression vector containing above-mentioned homologous recombination enzyme, it is characterised in that the carrier is with wide Build and obtain based on the replicon pBBR1 of host, include pBBR1 replication orgins, kalamycin resistance gene (km^R), sandlwood Sugared inducible promoter (P_Rha) and reached by rhamnose inducible promoter control table homologous recombination enzyme Red β -7029/Red α - 7029；- the Km of pBBR1-Rha-BA 7029 are named as, its nucleotide sequence is as shown in SEQ ID No.3.Recombination function albumen profit Induced expression is carried out with Rha inducible promoters.

Homologous recombination enzyme described in claim 1 of the present invention is with deriving from E.coli double-stranded DNA Exonucleolytic enzyme levels Albumen Red γ are incorporated in the application improved in Burkholderia In vivo recombination efficiency.

Wherein, the application process is：

(1) based on the replicon pBBR1 of wide host, structure includes pBBR1 replication orgins, kalamycin resistance gene (km^R), rhamnose inducible promoter (P_Rha) and reached by rhamnose inducible promoter control table recombinase Red γ- E.coli/Red β -7029/ Red α's -7029 carries outside the homologous recombination enzymes of Red α/Red β 7029 and E.coli double-stranded DNA nucleic acid Enzyme cutting suppresses albumen Red γ recombinase expression vector, is named as-the Km of pBBR1-Rha-RedG-BA 7029, its nucleotides sequence Row are as shown in SEQ ID No.4；

(2) to competent cell Optimization of preparation, the optimization of DNA inversion quantities, antibiotic selection concentration optimization, homologous brachium Degree optimization, it is determined that optimal condition of work is：Initial OD values are 0.1 bacterium solutions of DSM 7029, and 30 DEG C, 950rpm cultures 14 are small Shi Hou, add the 10mg ml of volume ratio 2%^-1Sandlwood sugar juice, 30 DEG C, 950rpm, induce 1.5h, with normal temperature it is double boil off from Sub- water process competent cell simultaneously carries out electric conversion in normal temperature；

(3) under the condition of work of optimization, expression vector is in (the Polyangium of Burkholderia DSM 7029 Brachysporum strain DSM 7029) in carry out gene substitution, gene is deleted with frame and gene insertion, using 50 ± The short homology arms of 10bp carry out a step knockout to a series of 100kb of the genomes of DSM 7029,200kb, 500kb fragments, obtain phase Answer a series of mutant strains；Pass through the measure to these mutant strain growth curves, it was demonstrated that large fragment is carried out to the genomes of DSM 7029 Knockout can significantly improve the growth rate and biomass of the mutant strain.

Specifically, Burkholderia homologous recombination enzyme of the present invention answering in the genome large fragments of DSM 7029 knockout With.

The present invention is using the homologous recombination enzymes of RedG-Red α/Red β 7029 with homology arm and apra^RThe PCR productions of gene Thing is respectively 100kb (3054675-3154840) and 200kb (3054675-3254511) to length on DSM7029 genomes Fragment be replaced.Construct DSM7029 Δ 100k mutant strains and DSM7029 Δ 200k mutant strains.Pass through and compare DSM7029 Δ 100k mutant strains and DSM7029 Δ 200k mutant strains and wild type DSM 7029 growth curve are found： The logarithmic phase duration of DSM7029 Δ 100k mutant strains and DSM7029 Δ 200k mutant strains and the maximum biology that can reach Measure the DSM 7029 apparently higher than wild type.

Application of the homologous recombination enzyme of the present invention in compound glidopeptin is obtained.

Wherein, the application process is：Using homologous recombination enzyme described in claim 1 to Burkholderia DSM 7029 The gene cluster cluster 6A of (Polyangium brachysporum strain DSM 7029) genome carry out promoter Insertion and knockout deactivation maneuver, build the mutant strains of DSM 7029, and compound glidopeptin is obtained by its tunning, its Concrete operation step and feature are as follows：

Promoter fragment of the both ends with 50bp homology arms is inserted into the bases of DSM 7029 first with Red α/Red β 7029 Because building DSM 7029P before cluster cluster 6A core gene_apraCluster 6A mutant strains, while utilize Red α/Red β 7029 is replaced with the gene cluster cluster 6A of resistant gene DSM 7029 of the both ends with 50bp homology arms nucleus The generation inactivation structure Δ cluster 6A mutant strains of DSM 7029, pass through HPLC comparative analysis DSM 7029P_apra cluster 6A With the Δ cluster 6A of DSM 7029 and wild type DSM 7029 fermentation crude extract, DSM 7029P are determined_apra Cluster 6A can produce a kind of new compound, and its charge-mass ratio is：M/z 676.2, [M+H]²⁺, the compound has been carried out point From purifying and structure elucidation, and it is glidopeptin by the Compound nomenclature.

Application of the homologous recombination enzyme of the present invention in compound rhizopeptin is obtained.

Wherein, application process is：It instead of respectively with resistant gene using RedG-Red α β -7029 homologous recombinations enzymes The Phosphopantetheinyl transferase gene (PPTase) of the bacterial strains of P.rhizoxinica HKI 454 (DSM 19002) and Rhizomycin biological synthesis gene cluster (rhiz) constructs the Δ PPTase mutant strains of DSM 19002 and DSM19002 Δs rhiz mutation Strain, tunning is analyzed by HPLC/MS and shown：Hair in the wild types of DSM 19002 and DSM19002 Δ rhiz mutant strains A metabolite in ferment product be present：m/z 732.38, [M+H]⁺, and in the Δ PPTase mutant strains of DSM 19002 this Metabolite disappears, and prompts the product that this metabolite is probably Non-ribosomal peptide approach or polyketide synthase approach, and The yield of the product is far above wild type in the mutant strain of rhizomycin synthetic gene cluster inactivation；It is homologous heavy using Red α β -7029 Group enzyme is found by DSM 19002 PKS/NRPS biological synthesis gene clusters knock out one by one：In to DSM 19002 The compound disappears after cluster 1 on the plasmid of source is knocked out；Composing type startup is further inserted before the gene cluster After son, it is found that the yield of the compound significantly improves, so that it is determined that the synthetic gene cluster of the compound, and pass through startup Son insertion improves the yield of the compound, and is named as rhizopeptin.

Compared with prior art, the present invention has following beneficial effect：The invention discloses one group of Burkholderia is homologous Recombinase-Red α/Red β 7029, this group of enzyme are to report first.The invention can effectively mediate non-compared with prior art The generation of homologous recombination in the Burkholderia body of Natural Transformation.The present invention answers this group of recombinase structure with wide host On system pBBR1 carrier, inducible promoter-rhamnose promoter (P is placed in_rha) control under enable this group of recombinase Enough applications controllable in a variety of Burkholderias.The present invention is realized to Burkholderia using the restructuring enzyme system of structure Polyangium brachysporum DSM 7029 and P. rhizoxinica HKI 454 genome are excavated, and are found that Two kinds of new compounds glidopeptin and rhizopeptin, both compounds are to report first.The present invention utilizes Red α/Red β 7029 complete the large fragment knockout to the genomes of DSM 7029, utilize the resistance with short homology arm (50bp) Genetic fragment realizes respectively to be knocked out to a step of 100kb and 200kb fragment on the genomes of DSM 7029, by bigger Fragment knockout mutant strain and and wild type DSM 7029 growth curve it was found that large fragment knock out mutant strain pair Number duration phase and the maximum biomass that can reach significantly improve.The present invention by newfound Red α/Red β 7029 with Double-stranded DNA Exonucleolytic enzyme level albumen Red γ from E.coli are used in combination with, and drastically increase this group of recombinase Operating efficiency.The discovery of the recombinase can largely promote excavation to the genome of Burkholderia, using and change Make, new Sample Storehouse can be provided for drug screening, had broad application prospects.

Brief description of the drawings

Fig. 1 (A) Burkholderia homologous recombination expression of enzymes carrier schematic diagram.

Wherein：Km：Kalamycin resistance gene；pBBR1:PBBR1 replicons；RhaR and rhaS rhamnose promoters are adjusted Gene； P_Rha：Rhamnose promoter；Red β -7029 and Red α -7029：Burkholderia homologous recombination enzyme；RedG:Escherichia coli Double-stranded DNA Exonucleolytic enzyme level albumen Red γ.(B) different length homology arm is compared in DSM7029 to recombination efficiency Influence.Wherein：gba:Recombinase combination from Escherichia coli bacteriophage lambda, the suppression of g- Escherichia coli double-stranded DNAs exonuclease The single-stranded Exonucleolytic of annealing albumen Red β, a- Escherichia coli bacteriophage lambda 5 ' -3 ' of albumen Red γ, b- Escherichia coli bacteriophage lambda processed Influence (D) of the different feeling state cell preparation temperature to recombination efficiency is compared in DSM7029 in enzyme Red α (C) DSM 7029 Influence of the different recombinase inducing temperatures to recombination efficiency.(E) in DSM 7029 comparative studies induction time to recombination efficiency Influence.(F) different competent cells is studied in DSM7029 and prepares influence of the buffer solution to recombination efficiency.Wherein：H： ddH₂O；S：10% sucrose solution；S+H：10%+2 μM of sucrose solution HEPS；G：10% glycerite；G+H：10% glycerine + 2 μM of HEPS of solution；(G) influence of the different competent cell concentration to recombination efficiency is studied in DSM7029.(H) work Condition optimizing is front and rear to utilize resistant gene apra of the Burkholderia homologous recombination enzyme with 80bp homology arms^RIn DSM7029 Knock out the comparison of glidobactin synthetic genes cluster (2675183-2696188) efficiency.Recon is in A Baila resistant panels Upper screening simultaneously counts, error line, SD；N=3.

Applications of Fig. 2 RedG-Red β/Red α 7029 in compound glidopeptin is obtained.

Wherein：(A) the gene cluster cluster 6A of DSM 7029 composition and activation in situ and the schematic diagram of inactivation.(B)DSM 7029 cluster 6A promoters insert bacterium colony PCR proof diagrams.M:DL 5000ladder.(C)DSM 7029cluster 6A Knock out bacterium colony PCR proof diagrams.M:DL 5000ladder.1-6A-Papra-check-1,2-6A-Papra-check-2,3- 6A-delet-check-1,4-6A- delet-check-2 (D) HPLC/MS analyzes the wild types of (BPC+All MS) DSM 7029 With mutant strain (DSM 7029 △ cluster 6A and DSM 7029P_apra- cluster 6A) methanolic extract.(E)DSM 7029 wild types and DSM7029P_apra- cluster 6A methanolic extract carries out toluene emulsification experiment.DSM 7029P_apra– cluster 6A:DSM 7029cluster 6A original position activated mutant strains, the Δ cluster 6A of DSM 7029:DSM 7029cluster 6A Inactivating mutations strains, DSM 7029wt:The wild-type strains of DSM 7029.

Application of Fig. 3 Burkholderia homologous recombination enzymes in compound rhizopeptin is obtained.

Wherein：(A) the gene cluster cluster 1 of DSM 19002 composition and activation in situ and the schematic diagram of inactivation.(B)DSM The promoters of 19002 cluster 1 are inserted with knocking out bacterium colony PCR proof diagrams.M:DL 5000ladder.C：(B) HPLC/MS is analyzed The methanolic extract of the wild types of (BPC+All MS) DSM 19002 and activation cluster 1 in situ.DSM 19002P_apra– cluster 1:The activated mutant strains in situ of DSM 19002cluster 1, the Δ cluster 1 of DSM 19002:DSM The Inactivating mutations strains of 19002cluster 1, the Δ rhiz of DSM 19002:The rhizomycin synthetic gene cluster knockout mutationss of DSM 19002 Strain, the Δ PPTase of DSM 19002:DSM19002 Phosphopantetheinyl transferase gene Inactivating mutations strains, DSM 19002ΔrhizΔcluster 1：DSM 19002cluster 1 and rhizomycin synthetic gene cluster Inactivating mutations strain, DSM 19002Δrhiz P_apra–cluster 1：The activation in situ of DSM 19002cluster 1 inactivates with rhizomycin synthetic gene cluster Mutant strain.

Application of Fig. 4 Burkholderia homologous recombination enzymes in the genome large fragments of DSM 7029 knockout.

Wherein：(A) Red α β 7029 resist in the apra that DSM7029 intermediaries conduction band has different length homology arm under optimum condition Property gene pairs large fragment knock out the result of (50kb-200kb).(B) the PC R the results that large fragment knocks out.Recon Ah Uncle draws and screens and count, error line, SD in resistant panel；N=3.(C) the Δ 200kb of DSM 7029 that large fragment obtains after knocking out Mutant strain, DSM7029+pBBR1-Rha-BA7029-Km and the growth curves of wild type DSM 7029 measure.Wherein： 100k- delet-check-1/100k-delet-check-2：100k knocks out detection primer；200k-delet-check-1/200k- delet-check-2：200k knocks out detection primer；CK：Blank control.

Fig. 5 Burkholderia homologous recombination enzymes and the Red γ protein expression vectors pBBR1-Rha- from Escherichia coli RedG-BA 7029-Km schematic diagrames.

Embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples, method is unless otherwise specified in embodiment Use conventional method, the reagent used unless otherwise specified use conventional commercial reagent or the examination configured according to a conventional method Agent.

Reagent and instrument：

Reagent is mainly molecular biology experiment reagent in the present embodiment, and single-stranded nucleotide is to give birth to work biotechnology from Shanghai Co., Ltd synthesizes, and restriction enzyme, archaeal dna polymerase and DNA marker come from biotech firm of New England (New England Biolabs), antibiotic is bought from hero company (Invitrogen), and Pu Luomaige Luciferase Assay Reagent box is purchased from general Luo Maige biological reagent companies.The Escherichia coli being related in invention：GB05-dir is purchased from German gene bridge company (GeneBridges) Burkholderia being related in the present invention：Burkholderiales strain DSM 7029, Burkholderia rhizoxinica HKI454, Burkholderia phytofirmans PsJN buy micro- from Germany Biological inoculum collection (DSMZ).

The preparation of the electric transformed cells recombinated：

Competent escherichia coli cell is prepared according to document report flow before.It is prepared by Burkholderia competent cell Method：The liquid CYMG culture mediums that there is suitable resistance equipped with 1.6ml are inoculated in yellow pipette tips picking single bacterium colony from flat board 2.0ml centrifuge tubes in, 30 DEG C, 950rpm, cultivate 24h, to OD600 5.0 or so；40 μ l are taken to be forwarded to new be equipped with In the 2.0ml centrifuge tubes of 1.6ml liquid CYMG culture mediums, 30 DEG C, 950rpm, 12-14h is cultivated, OD600 is reached 1.8 left It is right；, it is necessary to add corresponding derivant when being recombinated, 30 DEG C, 950rpm, 90min is induced, induces the expression of recombinase； 9300rpm centrifuges 1min, collects thalline, abandons supernatant.Add 1ml ddH₂O resuspension cells, 9700rpm centrifuge 1min again, Supernatant is abandoned, adds 1 ml ddH again₂Cell is resuspended in O, and 9700rpm centrifuges 1min, abandons supernatant, most cell is suspended at last again In 25 μ l or so water；1 μ g DNA are added, cell and DNA mixture are shifted in 1mm electricity revolving cups after mixing, 1350V electricity Hit；The LB culture mediums of 1ml antibiotic-frees are added into electric revolving cup, are blown and beaten repeatedly, suspended cell, then is transferred to 1.5ml punchings Centrifuge tube in, 30 DEG C of recovery 4h；The bacterium solution after appropriate recovery is taken, is applied on the CYMG flat boards containing corresponding antibiotic, After 30 DEG C of culture 3d, picking recon carries out bacterium colony PCR identifications.

The recons of DSM 7029 ferment and the extracting method of compound：

1. correct recon is inoculated in the 250ml triangular flasks containing 50ml CYMG culture mediums, 30 DEG C, 180rpm Overnight incubation.

2. the 15ml that transfers stays overnight bacterium (7 in the 5L triangular flasks containing 1.5L CYMG culture mediums (containing suitable antibiotic) Bottle), 30 DEG C, 160rpm cultures 1d.

3. every bottle of addition 30ml XAD16 resin, 30 DEG C, 160rpm cultures 3d.

4. thalline and resin are separated by mesh sieve, and resin is washed with distilled water and (gone as far as possible degerming several times Body), the moisture on resin is blotted by filter paper.

5. resin is poured into the 5L triangular flasks of new drying, 600ml methanol is added, 30 DEG C, 160rpm cultivates 1h.

6. take out triangular flask, and by 600ml methanol by filtering the method for (filter paper) by resin and separating methanol, and by first Alkoxide component is placed to be rotated on Rotary Evaporators.

7. 600ml methanol is added in the triangular flask containing resin, 30 DEG C, 160rpm cultures 1h.

8. step is the same as 6.

9. repeat step 5 and 6.

10. a pair methanol crude extract is weighed (2.4g), and carry out HPLC-MS detections.

HPLC-MS testing conditions：Column:2×100mm2.2μm

Rate:300μl/min

Sample:3μl

UV:254nm

A-H2O 0.1%FA

B-AcN 0.1%FA

0-3min 5%B

3-18min 5%-95%B

18-22min 5%B

22-25min 5%B

ESI+AutoMS2 100-1500

Precusor 2ions

Compound purification process：

1. the methanol crude extract by more than is dissolved and centrifuged with certain methanol, positive is added in methanol crude extract Silica gel, Rotary Evaporators are evaporated.

2. crude extract silica gel content is added to the elution separation that various concentrations gradient is carried out in normal phase silicagel column.(dichloro Methane, 20:1 dichloromethane and methanol, 15:1 dichloromethane and methanol, 10:1 dichloromethane and methanol, 5:The two of 1 Chloromethanes and methanol, 3:1 dichloromethane and methanol, 1:1 dichloromethane and methanol, methanol；Above solvent all adds formic acid)

3. after the different component of collection is evaporated by Rotary Evaporators, add the methanol (7-10ml) of certain volume It is distributed into after different component is dissolved in different test tubes, and carries out HPLC-MS detections.Testing conditions are same as above.

4. component 25 to component 45 is incorporated into a pipe, referred to as in component 1, rotation is evaporated and weighs (1.2g).

5. No. 1 is dissolved and centrifuged with the methanol of certain volume by component, a certain amount of reverse phase silica gel is added, rotation is evaporated.

6. the elution separation of progress various concentrations gradient in reverse phase silica gel post is added to regard to the reverse phase silica gel composition of crude extract. (20% methanol, 25% methanol, 30% methanol, 35% methanol, 40% methanol, 45% methanol, 50% methanol, 55% methanol, 60% Methanol+formic acid, 65% methanol+FA, 70% methanol+FA, 80% methanol+formic acid, 90% methanol+formic acid, 100% methanol+FA, 1:1 dichloromethane and methanol+formic acid.)

7. after the different component of collection is evaporated by Rotary Evaporators, add the methanol (7-10ml) of certain volume It is distributed into after different component is dissolved in different test tubes, and carries out HPLC-MS detections.Condition is same as above.

8. rotating and being evaporated component 90-1,90-2 respectively, and weigh, 90-1 (80mg), 90-2 (10mg).

9. by component 90-1, after 90-2 is dissolved with methanol, isolated and purified by semi-preparative HPLC.Separation Method：Component 90-1,90-2 (0-3min, 40%；3-10min, 40%-55%；10-23min, 55%-85%；23.1min 95%；23.1-27min 95%；27.1min 40%；27.1-31min, 40%) it is used for 3 compounds of purified pool, and steam It is dry.

10. the material isolated and purified is carried out into HPLC-MS detections respectively, NMR identifications are carried out after being then evaporated.

Embodiment 1：The optimization of the screening of Burkholderia homologous recombination enzyme, the structure of expression vector and condition of work is (real Apply the primer sequence that example is related to and see list)

(1) Burkholderia homologous recombination enzyme Red beta/alphas -7029 are obtained by bioinformatics and transcription group Analysis and Screening And the structure of recombinase expression vector.

Reported that the Red/ET recombinant techniques in Escherichia coli can not use in the Burkholderia of non-natural conversion before, Present invention demonstrates that the Red/ET recombinant techniques in Escherichia coli are unable to the Hes of high efficiency regulatory DSM 7029 on plasmid or genome The homologous recombination of other Natural Transformation Burkholderias.Genome in Burkholderia body is deleted and integration is still a problem, this It is the basic demand of exonuclease-recombinase to mean host specificity.The present invention analyzes the Hes of DSM 7029 by BLAST The hypothesis DNA recombinant proteins of other Burkholderias, the amino acid sequence using Red α β or recE/recT are standard in DSM The similar albumen of one group of Red is found that in 7029.Albumen similar this group of Red is Red beta/alphas -7029, Red in DSM 7029 α -7029 is exonuclease (similarity with bacteriophage lambda Red α is 28%)；Red β -7029 be bacteriophage recombinant protein (with 31%) bacteriophage lambda Red β similarity is.Red β -7029 and Red α -7029 gene number are AAW51_RS10365/ AAW51_RS10370.Based on this, the present invention proposes that can efficiently mediate the homologous heavy of Burkholderia In vivo recombination generation Group enzyme, it is characterised in that the homologous recombination enzyme includes an albumen wp_047194557.1 with exonuclease function, Red α 7029 and an albumen wp_053013463.1 with single-stranded annealing protein function are named as, is named as Red β 7029； Wherein：The mrna length of the Red α 7029 is 660 base-pairs, and its nucleotide sequence is as shown in SEQ ID No.1, the base Because encoding 219 amino acid encodings, shown in its amino acid sequence SEQ ID No.5；The mrna length of the Red β 7029 is 873 base-pairs, its nucleotide sequence is as shown in SEQ ID No.2,291 amino acid encodings of the gene code, its amino acid Shown in sequence SEQ ID No.6.The present invention also proposes a kind of expression vector containing above-mentioned homologous recombination enzyme, it is characterised in that institute It is to build to obtain based on the replicon pBBR1 of wide host to state carrier, includes pBBR1 replication orgins, kalamycin resistance base Because of (km^R), rhamnose inducible promoter (P_Rha) and the homologous recombination enzyme Red that is reached by rhamnose inducible promoter control table β-7029/Redα-7029；- the Km of pBBR1-Rha-BA 7029 are named as, its nucleotide sequence is as shown in SEQ ID No.3.

Recombination function plasmid is built based on the replicon pBBR1 of wide host, and recombination function albumen utilizes sandlwood Sugared promoter carries out induced expression.

The construction method of pBBR1-Rha-Red_gba-kan plasmids：PBBR1-Rha-GFP-kan is extracted from Escherichia coli Plasmid, 40ul plasmid NdeI digestion 3h, agarose gel electrophoresis 90V is taken to carry out race glue, until fragment is run to gel bottom The gel recovery of linear carrier is carried out, using pSC101-BAD-gba-tet plasmids as template, gba-3/gba-5 expands for primer Red_gba fragments, by the culture of GB-dir overnight bacterials and 30 DEG C of switching 1.3ml culture 2h, add 20ul arabinose derivants 37 DEG C of induction 40min, pBBR1-Rha-kan linear carriers and red_gba fragments 1350V electric shock corotation are dissolved into induction In GB05-dir, 37 DEG C of recovery 1h, 10000rpm, which centrifuges thalline and rule, is applied to the resistant panel of kanamycins 15,37 DEG C It is incubated overnight and chooses monoclonal and identified, correctly clone carries out bacterium guarantor.

The construction method of pBBR1-Rha-BA7029-Km plasmids：PBBR1-Rha-gba plasmids are extracted from Escherichia coli, 40ul plasmid restriction enzyme HindIII and NdeI digestion 3h, agarose gel electrophoresis 90V is taken to carry out race glue, until piece Section is run to the fragment of gel extraction 5084bp behind gel bottom, using the genomes of DSM 7029 as template, with primer pair 7029BA- 01A-1 and 7029BA-01A-2 obtains Red β/Red α -7029 genes by PCR, by the culture of GB-dir overnight bacterials and 30 DEG C Transfer 1.3ml culture 2h, adds 37 DEG C of induction 40min of 20ul arabinoses derivant, will return PCR primer and plasmid enzyme restriction Receive product 1350V electric shock corotation to dissolve into the GB05-dir of induction, 37 DEG C of recovery 1h, 10000rpm centrifugation thalline and painting of ruling Cloth is to the resistant panel of kanamycins 15, and 37 DEG C are incubated overnight and choose monoclonal and identified, correctly clone carries out bacterium guarantor.

The construction method of pBBR1-Rha-RedG-BA7029-Km plasmids：PBBR1-Rha-gba is extracted from Escherichia coli Plasmid, 40ul plasmid restriction enzyme HindIII and DraI digestion 3h, agarose gel electrophoresis 90V is taken to carry out race glue, Gel extraction 6892bp fragment, using the genomes of DSM 7029 as template, uses primer pair after fragment is run to gel bottom 7029BA-01B-1 and 7029BA-01A-2 obtains Red β/Red α -7029 genes by PCR, by GB-dir overnight bacterial cultures And 30 DEG C of switching 1.3ml culture 2h, 37 DEG C of induction 40min of 20ul arabinoses derivant are added, will be by PCR primer and plasmid Digestion recovery product 1350V electric shock corotation is dissolved into the GB05-dir of induction, and 37 DEG C of recovery 1h, 10000rpm centrifugation thalline are simultaneously Line is applied to the resistant panel of kanamycins 15, and 37 DEG C are incubated overnight and choose monoclonal and identified, correctly clone is carried out Bacterium protects.Obtain-the Km of pBBR1-Rha-RedG-BA 7029 (BA 7029:Redαβ7029).

(2) Burkholderia homologous recombination enzyme RedG-Red β α -7029 optimum combination system operating conditions in DSM 7029 Optimization

The present invention have studied homologous arm lengths and the different competent cell preparation methods that the DNA of conversion is carried respectively Influence to homologous recombination efficiency.

The influence of homologous arm lengths counterweight group is the A Baila by that will carry different length (50bp-100bp) homology arm Resistant gene PCR products are transferred in the DSM 7029 of the recombinases of induced expression Red α β 7029, to 21.2kb's Glidobactin biological synthesis gene clusters carry out part (2675183-2696188) and replaced.Using rk2-apra-km as template, Respectively with different primer pair 100-apra-glb-1/2,80-apra-glb-1/2,50-apra-glb-1/2 are expanded by PCR Increase and obtain the PCR primer with different length homology arm, then converted by electricity by PCR primer (1 μ of the gel extraction of equivalent G) it is transferred in the DSM 7029 induction of restructuring expression of enzymes, and in apra (20 μ g ml^-1) screening is carried out on CYMG flat boards turned Beggar, checking is carried out to transformant by bacterium colony PCR with primer pair glb-delet-check-1/2 respectively and obtains correctly weight Group.As a result the wild types of DSM 7029 are shown in and carry pBBR1-Rha-gba-km or pBBR1-Rha-Red G-km matter PBBR1-Rha-Red G-BA 7029-Km or pBBR1-Rha- are being carried in the DSM 7029 of grain without correct recon Homologous arm lengths increase (50bp~100bp) recombination efficiency improves (Figure 1B) in the DSM 7029 of BA 7029-Km plasmids.

The present invention also have detected influence of the different preparation methods of the competent cells of DSM 7029 to recombination efficiency.

Both ends are carried into 80bp and apra^RPCR primer be transferred to the DSM 7029 for expressing RedG-Red α/Red β 7029 In, 21.2 kb Glidobactin synthetic genes cluster (2675183-2696188) is replaced, respectively in room temperature (RT) or (OI) prepares competent cell to person on ice, as a result shows that the number that room temperature prepares the transformant that competent cell obtains is on ice Prepare five times (Fig. 1 C) of competent cell efficiency.

Present invention determine that influence of the different inducing temperatures and recovery temperature to recombination efficiency.

This part is still the apra that 80bp homology arms are carried with both ends^RResistance fragments have been knocked out on DSM7029 genomes Glidobactin synthetic genes cluster (2675183-2696188).Transfer after the 4h homogeneous OD600 values of bacterium solution, be divided into four groups, Induced and recovered at different temperature respectively, every group of three repetitions, 4 inducing temperatures：25℃、30℃、32℃、37 DEG C, induction time 1h.After induction terminates, carry out normal temperature electricity and turn, the recovery time after electricity turns is 3h, recovery temperature isogeneous induction Temperature, i.e., how many how many degree recoveries of degree induction, after recovery terminates, centrifugation bacterium solution abandons supernatant, and thalline is resuspended in into 100 microlitres After CYMG fluid nutrient mediums, full coat is in apra (20 μ g ml^-1) CYMG flat boards, optimal culture, induction are understood by the result It is 30 DEG C (Fig. 1 D) with recovery temperature.

Present invention determine that RedG-Red β/Red α -7029 most suitable induction time.

This part is still the apra that 80bp homology arms are carried with both ends^RResistance fragments have been knocked out on DSM7029 genomes Glidobactin synthetic genes cluster (2675183-2696188).Transfer after the 4h homogeneous OD600 values of bacterium solution, be divided into four groups, Induced and recovered at 30 DEG C respectively, every group of three repetitions, induction time is respectively：0.5h、1h、1.5h、2h.Induction knot Shu Hou, the OD values of bacterium solution when the OD values of all bacterium solutions are tuned into induction 0.5h, room temperature treatment competent cell and electricity turn, multiple After Soviet Union terminates, centrifugation bacterium solution abandons supernatant, and after thalline is resuspended in into 100 microlitres of CYMG fluid nutrient mediums, full coat is in apra (20 μ g ml^-1) CYMG flat boards, by the result understand optimal induction time be 1.5h (Fig. 1 E).Next present invention determine that most having Buffer solution is prepared beneficial to the competent cell of DSM7029 restructuring.Transfer after the 14h homogeneous OD600 values of bacterium solution, be divided into five groups, point Do not induced and recovered at 30 DEG C, every group of three repetitions, induction time 1.5h.Induce after terminating with 5 kinds of different impressions State prepares buffer solution room temperature treatment competent cell.5 kinds of competence prepare buffer solution and are respectively：ddH₂O, 10% sucrose is molten Liquid (S) ,+2 μM of HEPS of 10% sucrose solution (SH), 10% glycerite (G) ,+2 μM of HEPS of 10% glycerite (SH), 30 DEG C recovery 3h, after recovery terminates, after recovery terminates, centrifugation bacterium solution abandons supernatant, and thalline is resuspended in into 100 microlitres of CYMG liquid trainings After supporting base, full coat is in apra (20 μ g ml^-1) CYMG flat boards, by the part of test results we determined that prepared by optimal competence Buffer solution is ddH₂O (Fig. 1 F).When reporting that Escherichia coli and other Gram-negative bacterias entered exponential phase of growth before, outside The transformation efficiency of source DNA is highest, and the present invention has surveyed and drawn the DSM7029 bacterium for carrying the expression plasmid of Red γ-Red α/βs 7029 Growth curve of the strain at 30 DEG C, it is determined that competent cell concentration mediates the influence of restructuring to RedG- Red β/Red α -7029. The value of initial OD 600 of bacterium solution is 0.1, takes 1ml bacterium solutions every 2h, determines OD600 values.Bacterium gives birth in 12h into index after measured For a long time, we choose the measure for the thalline progress recombination efficiency for having cultivated 9h, 10h, 11h, 12h, 13h, 14h, 15h, it is determined that weight Group efficiency highest incubation time.Three repetitions of every group of incubation time, terminate since 9h to 15h, and one is taken every a hour It is the same that secondary sample, induction and electricity turn condition.OD values corresponding to 9h-15h are shown in broken line on Fig. 1 G, 30 DEG C of recovery 3h, recover after terminating, Apply apra (20 μ g ml^-1) CYMG flat boards, experimental result is shown in broken line under Fig. 1 G, it is determined that the Best Times of induction restructuring expression of enzymes Point is 14h (Fig. 1 G).

The optimization of condition is recombinated more than, present invention determine that in RedG-Red β/Red α -7029 mediation DSM7029 bodies The optimal conditions of restructuring：Initial OD values are 0.1 DSM7029 bacterium solutions, 30 DEG C, after 950rpm is cultivated 14 hours, add 2% Rhamnose (10 mg ml^-1), 30 DEG C, 950rpm, induce 1.5h, with the double distilled deionized water of normal temperature handle competent cell and Normal temperature carries out electric conversion.Using the condition of work after optimization, will can recombinate in the DSM7029 bodies that RedG-Red α/βs 7029 mediate The efficiency of restructuring improves three times (Fig. 1 H).

Embodiment 2：Application of the Burkholderia homologous recombination enzyme in compound glidopeptin is obtained.

The genetic manipulation system of structure can be used for exploring new secondary metabolite, and the present invention is based on biological information credit Analysis predicts the natural products biological synthesis gene cluster that several PKSs and NRPSs classes on the genomes of DSM 7029 be present： Cluser 6A (3068287-311501), cluster 7 (3161734-3234609) and (3950755- of cluster 11 4033151).Transcription group analyzes the core gene expression quantity of cluser 6A (3068287-3115010) under different condition all It is very low, knock out the LC-MS analysis results of strain zymotic fluid by comparing DSM7029 wild types and DSM 7029 and cluster 6A Fail to find corresponding secondary metabolite, therefore it is concluded that the two gene clusters are silences in wild type DSM7029 's.Started in present invention research using RedG-Red α/Red β 7029 by inserting composing type in cluster 6A (3067766) Sub- P_apraLine activating is entered to the gene cluster.Bioinformatic analysis prediction cluser 6A are made up of 5 genes, wherein three bases Because (AAW51 2719-AAW51 2721) coding Nonribosomal Peptide Synthetases synthesize the polypeptide of 12 amino acid；Gene AAW51 2717 encodes diaminobutyrate-2-oxoglutarate transaminases, acts on 2,4-diaminobutyric Acid biosynthesis；The predictive coding hydroxylases of Gene A AW51 2722；Gene A AW51 2723 encodes histidinol phosphatase.

By constitutive promoter (P in the present invention_apra) to carry out activation structure in situ prominent for insertion Gene A upstream (3067766) The P of mutant DSM 7029_apraCluster 6A, the present invention also utilize recombination system apra^RBase of the resistant gene to cluster6A The Δ cluster 6A (Fig. 2A) of mutant strain DSM 7029 are constructed because cluster has carried out part (3068779-3112764) knockout.It is first First, with primer pair 6A-P_apra- 1/2 obtains promoter fragment of the both ends with 50bp homology arms by PCR is inserted into and expresses Before the Gene A AW51 2717 of cluster 6A on Red γ-Red α/Red β 7029 DSM7029 genomes (3067766), And in apra (15 μ g ml^-1) screened on CYMG flat boards, and with primer pair 6A-P_apra- check-1/2 passes through bacterium colony PCR Transformant is verified.

The structure of the Δ cluster 6A mutant strains of DSM 7029, both ends are obtained with primer pair 6a-delet-1/2 by PCR Apra with 100bp homology arms^RGene pairs cluster 6A Partial Fragment (3069534-3111763) is replaced, and In apra (20 μ g ml^-1) screened on CYMG flat boards, and pass through PCR pairs of bacterium colony with primer pair 6A-delet-check-1/2 Transformant is verified.(Fig. 2 B-C) is through HPLC comparative analysis DSM 7029P_apraThe Δ of-cluster 6A and DSM 7029 Cluster 6A and wild type DSM 7029 fermentation crude extract, find DSM 7029P_apra- cluster 6A can produce 1 The new compound of kind, the charge-mass ratio of this compound are：(m/z 676.2, [M+H]²⁺) (Fig. 2 D).Cluser 6A module B Prediction is the starter unit of acyl group, it is meant that this gene cluster encodes a lipopeptid, and the lipopeptid of acellular toxin generally has table Face active function, biosurfactant can reduce the tension force of water/hydrophobic surface, and the present invention is proved by toluene emulsification experiment After the activation cluser 6A of original position, the fermentation extract of the mutant strain mixes with organic solvent can produce emulsification, and wild Emulsification can not then occur for raw type DSM7029 and knockout cluster6A DSM7029 mutant strains fermentation extract, it was demonstrated that Cluster6A is activated (Fig. 2 E).Cluser 6A product is named as glidopeptin by the present invention.

Embodiment 3：Application of the Burkholderia homologous recombination enzyme in compound rhizopeptin is obtained：

(1) structure of the Δ PPTase mutant strains of DSM 19002.Using rk2-apra-km plasmids as template, primer pair is used 19002-RhizKO-apra-1/2 is utilized apra of the both ends with 50bp homology arms by PCR^RResistant gene is to expressing Phosphopantetheinyl transferase (PPTase) base on Red γ-Red α/Red β 7029 DSM19002 genomes Because being substituted, and in apra (20 μ g ml^-1) screened on CYMG flat boards, and with primer pair 19002-PPTaseKO- Check-1/2 is verified by bacterium colony PCR to transformant, obtains the Δ PPTase mutant strains of DSM 19002.

(2) structure of the Δ rhiz mutant strains of DSM 19002, using rk2-apra-km plasmids as template, uses primer pair 19002-RhizKO-apra-1/2 is utilized apra of the both ends with 50bp homology arms by PCR^RResistant gene is to expressing Rhizoxin synthetic genes cluster on RedG-Red α/Red β 7029 DSM19002 genomes carries out part (1646431- 1647682) substitute, in apra (20 μ g ml^-1) screened on CYMG flat boards, with primer pair 19002-RhizKO-check- 1/2 is verified by bacterium colony PCR to transformant, obtains the Δ rhiz of mutant strain DSM 19002.Comparative analysis is detected through HPLC DSM 19002 Δ rhiz, DSM 19002 Δ PPTase and the strain DSMs 19002 of wild type P.rhizoxinica HKI 454 Ferment crude extract, in the fermentation of the Δ rhiz of DSM 19002 and the strain DSMs 19002 of wild type P.rhizoxinica HKI 454 It is found that a kind of new compound in thing, charge-mass ratio is m/z 732.3, [M+H]⁺, and the compound is in the Δs of DSM 19002 Yield in rhiz will be apparently higher than the yield (Fig. 3 C) in wild type P.rhizoxinica HKI 454.By the compound Disappearance in the Δ PPTase mutant strains of DSM 19002, it is PKS/NRPS class compounds to determine the compound, based on this in order to The synthetic gene cluster of the compound is determined, we are to 11 PKS/NRPS classes on the genomes of DSM 19002 and indigenous plasmid Synthetic gene cluster has been carried out knocking out inactivation and promoter insertion activation one by one, and LC-MS analyses are carried out to zymotic fluid, final true The product that the fixed compound is the cluster 1 (plasmid 1,3940-66916) on the indigenous plasmids of DSM 19002. First, promoter fragment of the both ends with 50bp homology arms is obtained by PCR with primer pair C1-Papra-1/2 and is inserted into expression Before cluster 1 on RedG-Red α/Red β 7029 DSM7029 genomes (19,923-20,001) (Fig. 3 A), and apra(20μg ml^-1) screened on CYMG flat boards, and with primer pair C1-Papra-check1/2 by bacterium colony PCR to turning Beggar is verified, obtains mutant strain DSM 7029P_apra-cluster 1。

The structure of the mutant strains of 19002 Δ cluster of DSM 1, both ends are obtained by PCR with primer pair C1KO-apra-1/2 Apra with 100 bp homology arms^RGene pairs cluster 1 Partial Fragment (21,439-22,629) is replaced (figure 3A), and in apra (20 μ g ml^-1) screened on CYMG flat boards, and pass through bacterium colony with primer pair C1-delet-check-1/2 PCR is verified (Fig. 3 B) to transformant.Find to insert constitutive promoter before cluster1 through HPLC detection comparative analysis The yield (Fig. 3 D) of the compound can significantly be improved.

Embodiment 4：Application of the Burkholderia homologous recombination enzyme in the genome large fragments of DSM 7029 knockout.

On the basis of condition optimizing, respectively with different primer pair 100-100k-delet-1/2,100-50k- Delet-1/2,100-200k-delet-1/2,80-100k-delet-1/2,80-50k-delet-1/2,80-200k- Delet-1/2,50-50k-delet-1/2,50-100k-delet-1/2,50-200k-delet-1/2 are expanded by PCR To the apra with different length homology arm^RPCR primer Red γ-Red α β 7029 bacterial strains of DSM 7029 in induced expression In the larger sequence fragment (3054675-3111708,3054675-3154840,3054675-3254511) on genome is struck Removing, the total length of target area is about 210kb (3054675-3111708), and in apra (20 μ g ml^-1) CYMG flat boards are enterprising Row screening obtains transformant, respectively with primer pair 100k-delet-check-1/2,200k-delet-check-1/2, Inside apra-1/2 carry out bacterium colony PCR checkings to transformant and obtain correct recon.By expressing Red γ-Red α β 7029, the genome sequence of large fragment can knock out (Fig. 4 A) by effective, and step restructuring is all just by bacterium colony PCR checkings True (Fig. 4 B).By determining the Δ 200k of mutant strain DSM 7029 and wild type DSM 7029 of large fragment knockout and carrying The DSM 7029 of recombinase expression plasmid:DSM7029+pBBR1-Rha-RedG-BA 7029-Km growth curve is surveyed It is fixed, as a result show that carrying out large fragment knocks out the Δ 200k of mutant strain DSM 7029 obtained exponential phase and can reach Maximum biomass all has be significantly improved (Fig. 4 C).

Subordinate list：The primer of design of the embodiment of the present invention and its sequence table

Sequence table

<110>Shandong University

<120>One group of Burkholderia homologous recombination enzyme and its expression vector and application

<141>2017-08-5

<160> 7

<210> 1

<211> 663

<212> DNA

<213> Polyangium brachysporum DSM 7029

<221>The exonuclease Red of Burkholderia 5 '-3 ' α from Polyangium brachysporum DSM 7029- 7029

<222>（1）…（663）

<400>1

atgatcgtct acaccgatcc ccaaggctcg ctcgaatggc tcgccgcccg gcgcggagtg 60

atcaccggat cacgcttcaa ggactgccgc gactacaacc agccgaccgc ggccgagaaa 120

aaggccggcg agacccgcgg caagccttcg aagacgctgc ttgcgtatgc catggacgtg 180

gcgcgcgagc gggtaggcgg cattgcgccg tcgaagttcg tgacccatgc catgcgcgcc 240

ggcacggagg aagagccgcc cgcacggatc gcgtatgagg ccgagaccgg ccatctggtc 300

gaggaagctg gcttcatcac gaccgacgac cggctcttcg gttgctcagt tgatggcttc 360

gtcggcgccg acggagtcat cgagatcaag acgatggtgt cgtccgacac cctcttccgc 420

gccgtcgtcc agggcgacat tagcgaatac atcgaccaga tcaacgggga aatgtggctg 480

ctcggccgca agtgggtcga cctcgtgctg tgggcgccgg atctggagcc gctcggcaag 540

cacctgacga ttcgacgcgt cgtgcgcgac gaggcagcca tccaagctct cgaagacgac 600

ctcatgacct ttgcccgact ggtcgctcag ttcgagaagg aactgcggaa ggaggcagca 660

tga 663

<210> 2

<211>876

<212> DNA

<213> Polyangium brachysporum DSM 7029

<221>The single-stranded annealing albumen Red β -7029 of Burkholderia from Polyangium brachysporum DSM 7029

<222>（1）…（876）

<400>2

atgaccaacg ccctcacgaa acaagagggc ggcgcactcg cgctgtccga ggctgaactg 60

ctgaacgtgc tcgcctcgtc gctgtacccg ggcgccgcgc ccgagtcgat caagctcgta 120

atcggctact gcaaggcagc gggcctcgac ccgatgcaaa agccggtgca catcgttccc 180

atgtgggacg gcaaggccaa gcggatgcgt gacgtggtga tgccaggcat caacctgtat 240

cgcacccagg ctgcgagatc cggccagttc gccggcatgt cggagcccga gttcgggcct 300

gacgtaacgc agaacgtcgg cggggtgacg atcactttcc cggactggtg ccgggtgact 360

gtgaaacgcg cgctagctga tggccgcatc gcggagttca ccgcgcgcga gtattggatc 420

gagaactacg cggtgaaggg tgggcaggag aagtcgacgg cacccaacgc catgtggcag 480

aagcggccac gcgggcagct tgcgaagtgc gcggccgcac aggctctgcg catcgccttc 540

cccgagatcg catcgcaagc gaccgcggaa gagatggagg gcaaatcgct gcagagcgac 600

gagccgctcg atccaccgcc ctcgcaggtc ccggccgaac tggtcgaggc agcgaaagcc 660

gctgcctcga agggggtgca agcctaccag gagttctggc aagccaccgg cgctgcaaac 720

cgcaaactgc tcgcgcccca ccacgcgagc ctcaaggagc aggcggcaaa ggccgacgcc 780

gaccgaacga tcgacgcgca gacccgggat ggtgcgggca gcgatggcgg caacgacgtc 840

ggcgacgaga tcgaagccac ggaggatcgg gaatga 876

<210> 3

<211> 7894

<212> DNA

<213>Artificial sequence

<221>Burkholderia homologous recombination expression of enzymes carrier pBBR1-Rha-BA 7029-Km

<222>（1）…（7894）

<400>3

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180

aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 240

cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 300

tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 360

gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 420

aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 480

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 540

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 600

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 660

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 720

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 780

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 840

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 900

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 960

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 1020

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1080

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1140

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1200

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 1260

ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 1320

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 1380

atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta 1440

tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 1500

tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatgca ttaatctttc 1560

tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt aaacaccacc 1620

gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat taacaggcgg 1680

ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata ttgattgatg 1740

gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca cgatgcctca 1800

tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg ggtaatcagc 1860

ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta acgatggcga 1920

ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc aaacggcaca 1980

tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc catccccatg 2040

ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat cgccccctgc 2100

cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa aaccagatcg 2160

ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc gccacgggta 2220

atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac aatcaccagc 2280

tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc agccacagcg 2340

actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg atgcgccacc 2400

gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac aaatacgttg 2460

agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac gcggtgacca 2520

gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc cacagcgata 2580

ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt tcatcagtcg 2640

caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct gccgatgtag 2700

cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct catcggcaaa 2760

atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt ccaggttctc 2820

ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct cgcgactggc 2880

ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca gctgtcgcac 2940

ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat ccagctcttg 3000

tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc gatacagcac 3060

attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat cgcgtacgaa 3120

acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa tacccgtgcc 3180

atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat ccgcctgcgg 3240

gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca cactatgtaa 3300

tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca cgaggtcagg 3360

ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt caagatacag 3420

cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa attgtgaaca 3480

tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt aacgagaagg 3540

tcgcgaattc aggcgctttt tagactggtc gtaatgaaca attcttaaga aggagatagt 3600

atacatgacc aacgccctca cgaaacaaga gggcggcgca ctcgcgctgt ccgaggctga 3660

actgctgaac gtgctcgcct cgtcgctgta cccgggcgcc gcgcccgagt cgatcaagct 3720

cgtaatcggc tactgcaagg cagcgggcct cgacccgatg caaaagccgg tgcacatcgt 3780

tcccatgtgg gacggcaagg ccaagcggat gcgtgacgtg gtgatgccag gcatcaacct 3840

gtatcgcacc caggctgcga gatccggcca gttcgccggc atgtcggagc ccgagttcgg 3900

gcctgacgta acgcagaacg tcggcggggt gacgatcact ttcccggact ggtgccgggt 3960

gactgtgaaa cgcgcgctag ctgatggccg catcgcggag ttcaccgcgc gcgagtattg 4020

gatcgagaac tacgcggtga agggtgggca ggagaagtcg acggcaccca acgccatgtg 4080

gcagaagcgg ccacgcgggc agcttgcgaa gtgcgcggcc gcacaggctc tgcgcatcgc 4140

cttccccgag atcgcatcgc aagcgaccgc ggaagagatg gagggcaaat cgctgcagag 4200

cgacgagccg ctcgatccac cgccctcgca ggtcccggcc gaactggtcg aggcagcgaa 4260

agccgctgcc tcgaaggggg tgcaagccta ccaggagttc tggcaagcca ccggcgctgc 4320

aaaccgcaaa ctgctcgcgc cccaccacgc gagcctcaag gagcaggcgg caaaggccga 4380

cgccgaccga acgatcgacg cgcagacccg ggatggtgcg ggcagcgatg gcggcaacga 4440

cgtcggcgac gagatcgaag ccacggagga tcgggaatga tcgtctacac cgatccccaa 4500

ggctcgctcg aatggctcgc cgcccggcgc ggagtgatca ccggatcacg cttcaaggac 4560

tgccgcgact acaaccagcc gaccgcggcc gagaaaaagg ccggcgagac ccgcggcaag 4620

ccttcgaaga cgctgcttgc gtatgccatg gacgtggcgc gcgagcgggt aggcggcatt 4680

gcgccgtcga agttcgtgac ccatgccatg cgcgccggca cggaggaaga gccgcccgca 4740

cggatcgcgt atgaggccga gaccggccat ctggtcgagg aagctggctt catcacgacc 4800

gacgaccggc tcttcggttg ctcagttgat ggcttcgtcg gcgccgacgg agtcatcgag 4860

atcaagacga tggtgtcgtc cgacaccctc ttccgcgccg tcgtccaggg cgacattagc 4920

gaatacatcg accagatcaa cggggaaatg tggctgctcg gccgcaagtg ggtcgacctc 4980

gtgctgtggg cgccggatct ggagccgctc ggcaagcacc tgacgattcg acgcgtcgtg 5040

cgcgacgagg cagccatcca agctctcgaa gacgacctca tgacctttgc ccgactggtc 5100

gctcagttcg agaaggaact gcggaaggag gcagcatgag tatacattga ctaccggaag 5160

cagtgtgacc gtgtgcttct caaatgcctg aggccagttt gctcaggctc tccccgtgga 5220

ggtaataatt gacgatatga tcatttattc tgcctcccag agcctgataa aaacggtgaa 5280

tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg 5340

cgacgcaacg cggggaggca gacaaggtat agggcggcga ggcggctaca gccgatagtc 5400

tggaacagcg cacttacggg ttgctgcgca acccaagtgc taccggcgcg gcagcgtgac 5460

ccgtgtcggc ggctccaacg gctcgccatc gtccagaaaa cacggctcat cgggcatcgg 5520

caggcgctgc tgcccgcgcc gttcccattc ctccgtttcg gtcaaggctg gcaggtctgg 5580

ttccatgccc ggaatgccgg gctggctggg cggctcctcg ccggggccgg tcggtagttg 5640

ctgctcgccc ggatacaggg tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc 5700

gtcctggtcg tcgtgatcaa ccaccacggc ggcactgaac accgacaggc gcaactggtc 5760

gcggggctgg ccccacgcca cgcggtcatt gaccacgtag gccgacacgg tgccggggcc 5820

gttgagcttc acgacggaga tccagcgctc ggccaccaag tccttgactg cgtattggac 5880

cgtccgcaaa gaacgtccga tgagcttgga aagtgtcttc tggctgacca ccacggcgtt 5940

ctggtggccc atctgcgcca cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc 6000

aataagcccg gcccacgcct catgcgcttt gcgttccgtt tgcacccagt gaccgggctt 6060

gttcttggct tgaatgccga tttctctgga ctgcgtggcc atgcttatct ccatgcggta 6120

gggtgccgca cggttgcggc accatgcgca atcagctgca acttttcggc agcgcgacaa 6180

caattatgcg ttgcgtaaaa gtggcagtca attacagatt ttctttaacc tacgcaatga 6240

gctattgcgg ggggtgccgc aatgagctgt tgcgtacccc ccttttttaa gttgttgatt 6300

tttaagtctt tcgcatttcg ccctatatct agttctttgg tgcccaaaga agggcacccc 6360

tgcggggttc ccccacgcct tcggcgcggc tccccctccg gcaaaaagtg gcccctccgg 6420

ggcttgttga tcgactgcgc ggccttcggc cttgcccaag gtggcgctgc ccccttggaa 6480

cccccgcact cgccgccgtg aggctcgggg ggcaggcggg cgggcttcgc cttcgactgc 6540

ccccactcgc ataggcttgg gtcgttccag gcgcgtcaag gccaagccgc tgcgcggtcg 6600

ctgcgcgagc cttgacccgc cttccacttg gtgtccaacc ggcaagcgaa gcgcgcaggc 6660

cgcaggccgg aggcttttcc ccagagaaaa ttaaaaaaat tgatggggca aggccgcagg 6720

ccgcgcagtt ggagccggtg ggtatgtggt cgaaggctgg gtagccggtg ggcaatccct 6780

gtggtcaagc tcgtgggcag gcgcagcctg tccatcagct tgtccagcag ggttgtccac 6840

gggccgagcg aagcgagcca gccggtggcc gctcgcggcc atcgtccaca tatccacggg 6900

ctggcaaggg agcgcagcga ccgcgcaggg cgaagcccgg agagcaagcc cgtagggcgc 6960

cgcagccgcc gtaggcggtc acgactttgc gaagcaaagt ctagtgagta tactcaagca 7020

ttgagtggcc cgccggaggc accgccttgc gctgcccccg tcgagccggt tggacaccaa 7080

aagggagggg caggcatggc ggcatacgcg atcatgcgat gcaagaagct ggcgaaaatg 7140

ggcaacgtgg cggccagtct caagcacgcc taccgcgagc gcgagacgcc caacgctgac 7200

gccagcagga cgccagagaa cgagcactgg gcggccagca gcaccgatga agcgatgggc 7260

cgactgcgcg agttgctgcc agagaagcgg cgcaaggacg ctgtgttggc ggtcgagtac 7320

gtcatgacgg ccagcccgga atggtggaag tcggccagcc aagaacagca ggcggcgttc 7380

ttcgagaagg cgcacaagtg gctggcggac aagtacgggg cggatcgcat cgtgacggcc 7440

agcatccacc gtgacgaaac cagcccgcac atgaccgcgt tcgtggtgcc gctgacgcag 7500

gacggcaggc tgtcggccaa ggagttcatc ggcaacaaag cgcagatgac ccgcgaccag 7560

accacgtttg cggccgctgt ggccgatcta gggctgcaac ggggcatcga gggcagcaag 7620

gcacgtcaca cgcgcattca ggcgttctac gaggccctgg agcggccacc agtgggccac 7680

gtcaccatca gcccgcaagc ggtcgagcca cgcgcctatg caccgcaggg attggccgaa 7740

aagctgggaa tctcaaagcg cgttgagacg ccggaagccg tggccgaccg gctgacaaaa 7800

gcggttcggc aggggtatga gcctgcccta caggccgccg caggagcgcg tgagatgcgc 7860

aagaaggccg atcaagccca agagacggcc cgag 7894

<210> 4

<211> 8328

<212> DNA

<213>Artificial sequence

<221>Burkholderia homologous recombination expression of enzymes carrier pBBR1-Rha-RedG-BA 7029-Km

<222>（1）…（8328）

<400> 4

atgaccaacg ccctcacgaa acaagagggc ggcgcactcg cgctgtccga ggctgaactg 60

ctgaacgtgc tcgcctcgtc gctgtacccg ggcgccgcgc ccgagtcgat caagctcgta 120

atcggctact gcaaggcagc gggcctcgac ccgatgcaaa agccggtgca catcgttccc 180

atgtgggacg gcaaggccaa gcggatgcgt gacgtggtga tgccaggcat caacctgtat 240

cgcacccagg ctgcgagatc cggccagttc gccggcatgt cggagcccga gttcgggcct 300

gacgtaacgc agaacgtcgg cggggtgacg atcactttcc cggactggtg ccgggtgact 360

gtgaaacgcg cgctagctga tggccgcatc gcggagttca ccgcgcgcga gtattggatc 420

gagaactacg cggtgaaggg tgggcaggag aagtcgacgg cacccaacgc catgtggcag 480

aagcggccac gcgggcagct tgcgaagtgc gcggccgcac aggctctgcg catcgccttc 540

cccgagatcg catcgcaagc gaccgcggaa gagatggagg gcaaatcgct gcagagcgac 600

gagccgctcg atccaccgcc ctcgcaggtc ccggccgaac tggtcgaggc agcgaaagcc 660

gctgcctcga agggggtgca agcctaccag gagttctggc aagccaccgg cgctgcaaac 720

cgcaaactgc tcgcgcccca ccacgcgagc ctcaaggagc aggcggcaaa ggccgacgcc 780

gaccgaacga tcgacgcgca gacccgggat ggtgcgggca gcgatggcgg caacgacgtc 840

ggcgacgaga tcgaagccac ggaggatcgg gaatgatcgt ctacaccgat ccccaaggct 900

cgctcgaatg gctcgccgcc cggcgcggag tgatcaccgg atcacgcttc aaggactgcc 960

gcgactacaa ccagccgacc gcggccgaga aaaaggccgg cgagacccgc ggcaagcctt 1020

cgaagacgct gcttgcgtat gccatggacg tggcgcgcga gcgggtaggc ggcattgcgc 1080

cgtcgaagtt cgtgacccat gccatgcgcg ccggcacgga ggaagagccg cccgcacgga 1140

tcgcgtatga ggccgagacc ggccatctgg tcgaggaagc tggcttcatc acgaccgacg 1200

accggctctt cggttgctca gttgatggct tcgtcggcgc cgacggagtc atcgagatca 1260

agacgatggt gtcgtccgac accctcttcc gcgccgtcgt ccagggcgac attagcgaat 1320

acatcgacca gatcaacggg gaaatgtggc tgctcggccg caagtgggtc gacctcgtgc 1380

tgtgggcgcc ggatctggag ccgctcggca agcacctgac gattcgacgc gtcgtgcgcg 1440

acgaggcagc catccaagct ctcgaagacg acctcatgac ctttgcccga ctggtcgctc 1500

agttcgagaa ggaactgcgg aaggaggcag catgagtata cattgactac cggaagcagt 1560

gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc cgtggaggta 1620

ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac ggtgaatccg 1680

ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac 1740

gcaacgcggg gaggcagaca aggtataggg cggcgaggcg gctacagccg atagtctgga 1800

acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt 1860

gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg catcggcagg 1920

cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag gtctggttcc 1980

atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc 2040

tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag cgattcgtcc 2100

tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg 2160

ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc ggggccgttg 2220

agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta ttggaccgtc 2280

cgcaaagaac gtccgatgag cttggaaagt gtcttctggc tgaccaccac ggcgttctgg 2340

tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt cctcgcaata 2400

agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc gggcttgttc 2460

ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat gcggtagggt 2520

gccgcacggt tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat 2580

tatgcgttgc gtaaaagtgg cagtcaatta cagattttct ttaacctacg caatgagcta 2640

ttgcgggggg tgccgcaatg agctgttgcg tacccccctt ttttaagttg ttgattttta 2700

agtctttcgc atttcgccct atatctagtt ctttggtgcc caaagaaggg cacccctgcg 2760

gggttccccc acgccttcgg cgcggctccc cctccggcaa aaagtggccc ctccggggct 2820

tgttgatcga ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc ttggaacccc 2880

cgcactcgcc gccgtgaggc tcggggggca ggcgggcggg cttcgccttc gactgccccc 2940

actcgcatag gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg cggtcgctgc 3000

gcgagccttg acccgccttc cacttggtgt ccaaccggca agcgaagcgc gcaggccgca 3060

ggccggaggc ttttccccag agaaaattaa aaaaattgat ggggcaaggc cgcaggccgc 3120

gcagttggag ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca atccctgtgg 3180

tcaagctcgt gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt gtccacgggc 3240

cgagcgaagc gagccagccg gtggccgctc gcggccatcg tccacatatc cacgggctgg 3300

caagggagcg cagcgaccgc gcagggcgaa gcccggagag caagcccgta gggcgccgca 3360

gccgccgtag gcggtcacga ctttgcgaag caaagtctag tgagtatact caagcattga 3420

gtggcccgcc ggaggcaccg ccttgcgctg cccccgtcga gccggttgga caccaaaagg 3480

gaggggcagg catggcggca tacgcgatca tgcgatgcaa gaagctggcg aaaatgggca 3540

acgtggcggc cagtctcaag cacgcctacc gcgagcgcga gacgcccaac gctgacgcca 3600

gcaggacgcc agagaacgag cactgggcgg ccagcagcac cgatgaagcg atgggccgac 3660

tgcgcgagtt gctgccagag aagcggcgca aggacgctgt gttggcggtc gagtacgtca 3720

tgacggccag cccggaatgg tggaagtcgg ccagccaaga acagcaggcg gcgttcttcg 3780

agaaggcgca caagtggctg gcggacaagt acggggcgga tcgcatcgtg acggccagca 3840

tccaccgtga cgaaaccagc ccgcacatga ccgcgttcgt ggtgccgctg acgcaggacg 3900

gcaggctgtc ggccaaggag ttcatcggca acaaagcgca gatgacccgc gaccagacca 3960

cgtttgcggc cgctgtggcc gatctagggc tgcaacgggg catcgagggc agcaaggcac 4020

gtcacacgcg cattcaggcg ttctacgagg ccctggagcg gccaccagtg ggccacgtca 4080

ccatcagccc gcaagcggtc gagccacgcg cctatgcacc gcagggattg gccgaaaagc 4140

tgggaatctc aaagcgcgtt gagacgccgg aagccgtggc cgaccggctg acaaaagcgg 4200

ttcggcaggg gtatgagcct gccctacagg ccgccgcagg agcgcgtgag atgcgcaaga 4260

aggccgatca agcccaagag acggcccgag accttcggga gcgcctgaag cccgttctgg 4320

acgccctggg gccgttgaat cgggatatgc aggccaaggc cgccgcgatc atcaaggccg 4380

tgggcgaaaa gctgctgacg gaacagcggg aagtccagcg ccagaaacag gcccagcgcc 4440

agcaggaacg cgggcgcgca catttccccg aaaagtgcca cctgggatga atgtcagcta 4500

ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg 4560

gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc 4620

agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt 4680

gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc 4740

gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 4800

gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 4860

gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 4920

tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 4980

agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 5040

ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 5100

tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 5160

acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 5220

ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 5280

gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 5340

ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 5400

tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 5460

ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 5520

ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 5580

cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 5640

ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 5700

ttcttcgccc acccccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct 5760

ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga 5820

attacaacag tttttatgca ttaatctttc tgcgaattga gatgacgcca ctggctgggc 5880

gtcatcccgg tttcccgggt aaacaccacc gaaaaatagt tactatcttc aaagccacat 5940

tcggtcgaaa tatcactgat taacaggcgg ctatgctgga gaagatattg cgcatgacac 6000

actctgacct gtcgcagata ttgattgatg gtcattccag tctgctggcg aaattgctga 6060

cgcaaaacgc gctcactgca cgatgcctca tcacaaaatt tatccagcgc aaagggactt 6120

ttcaggctag ccgccagccg ggtaatcagc ttatccagca acgtttcgct ggatgttggc 6180

ggcaacgaat cactggtgta acgatggcga ttcagcaaca tcaccaactg cccgaacagc 6240

aactcagcca tttcgttagc aaacggcaca tgctgactac tttcatgctc aagctgaccg 6300

ataacctgcc gcgcctgcgc catccccatg ctacctaagc gccagtgtgg ttgccctgcg 6360

ctggcgttaa atcccggaat cgccccctgc cagtcaagat tcagcttcag acgctccggg 6420

caataaataa tattctgcaa aaccagatcg ttaacggaag cgtaggagtg tttatcgtca 6480

gcatgaatgt aaaagagatc gccacgggta atgcgataag ggcgatcgtt gagtacatgc 6540

aggccattac cgcgccagac aatcaccagc tcacaaaaat catgtgtatg ttcagcaaag 6600

acatcttgcg gataacggtc agccacagcg actgcctgct ggtcgctggc aaaaaaatca 6660

tctttgagaa gttttaactg atgcgccacc gtggctacct cggccagaga acgaagttga 6720

ttattcgcaa tatggcgtac aaatacgttg agaagattcg cgttattgca gaaagccatc 6780

ccgtccctgg cgaatatcac gcggtgacca gttaaactct cggcgaaaaa gcgtcgaaaa 6840

gtggttactg tcgctgaatc cacagcgata ggcgatgtca gtaacgctgg cctcgctgtg 6900

gcgtagcaga tgtcgggctt tcatcagtcg caggcggttc aggtatcgct gaggcgtcag 6960

tcccgtttgc tgcttaagct gccgatgtag cgtacgcagt gaaagagaaa attgatccgc 7020

cacggcatcc caattcacct catcggcaaa atggtcctcc agccaggcca gaagcaagtt 7080

gagacgtgat gcgctgtttt ccaggttctc ctgcaaactg cttttacgca gcaagagcag 7140

taattgcata aacaagatct cgcgactggc ggtcgagggt aaatcatttt ccccttcctg 7200

ctgttccatc tgtgcaacca gctgtcgcac ctgctgcaat acgctgtggt taacgcgcca 7260

gtgagacgga tactgcccat ccagctcttg tggcagcaac tgattcagcc cggcgagaaa 7320

ctgaaatcga tccggcgagc gatacagcac attggtcaga cacagattat cggtatgttc 7380

atacagatgc cgatcatgat cgcgtacgaa acagaccgtg ccaccggtga tggtataggg 7440

ctgcccatta aacacatgaa tacccgtgcc atgttcgaca atcacaattt catgaaaatc 7500

atgatgatgt tcaggaaaat ccgcctgcgg gagccggggt tctatcgcca cggacgcgtt 7560

accagacgga aaaaaatcca cactatgtaa tacggtcata ctggcctcct gatgtcgtca 7620

acacggcgaa atagtaatca cgaggtcagg ttcttacctt aaattttcga cggaaaacca 7680

cgtaaaaaac gtcgattttt caagatacag cgtgaatttt caggaaatgc ggtgagcatc 7740

acatcaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc tggttgccaa 7800

tggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt tagactggtc 7860

gtaatgaaca attcttaaga aggagatata catatggata ttaatactga aactgagatc 7920

aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc atttcgcggg 7980

cgatattttc acagctattt caggagttca gccatgaacg cttattacat tcaggatcgt 8040

cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga gaaagaggca 8100

gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc gctatgcatc 8160

gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt tgatgacgat 8220

gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac cattgctcac 8280

caccaggttg atattgattc agaggtataa aacgagagga gggtatac 8328

<210> 5

<211> 220

<212> PRT

<213> Polyangium brachysporum DSM 7029

<221>The exonuclease Red of Burkholderia 5 '-3 ' α from Polyangium brachysporum DSM 7029- 7029 amino acid sequences

<222>（1）…（220）

<400> 5

MIVYTDPQGS LEWLAARRGV ITGSRFKDCR DYNQPTAAEK KAGETRGKPS KTLLAYAMDV 60

ARERVGGIAP SKFVTHAMRA GTEEEPPARI AYEAETGHLV EEAGFITTDD RLFGCSVDGF 120

VGADGVIEIK TMVSSDTLFR AVVQGDISEY IDQINGEMWL LGRKWVDLVL WAPDLEPLGK 180

HLTIRRVVRD EAAIQALEDD LMTFARLVAQ FEKELRKEAA 220

<210> 6

<211> 291

<212> PRT

<213> Polyangium brachysporum DSM 7029

<221>The single-stranded annealing albumen Red β -7029 of Burkholderia from Polyangium brachysporum DSM 7029 Amino acid sequence

<222>（1）…（291）

<400> 6

MTNALTKQEG GALALSEAEL LNVLASSLYP GAAPESIKLV IGYCKAAGLD PMQKPVHIVP 60

MWDGKAKRMR DVVMPGINLY RTQAARSGQF AGMSEPEFGP DVTQNVGGVT ITFPDWCRVT 120

VKRALADGRI AEFTAREYWI ENYAVKGGQE KSTAPNAMWQ KRPRGQLAKC AAAQALRIAF 180

PEIASQATAE EMEGKSLQSD EPLDPPPSQV PAELVEAAKA AASKGVQAYQ EFWQATGAAN 240

RKLLAPHHAS LKEQAAKADA DRTIDAQTRD GAGSDGGNDV GDEIEATEDR E 291

Claims (8)

1. one group can efficiently mediate the homologous recombination enzyme that Burkholderia In vivo recombination occurs, including one has exonuclease The albumen wp_047194557.1 of function, it is named as Red α 7029 and an albumen wp_ with single-stranded annealing protein function 053013464.1, it is named as Red β 7029；Wherein：The mrna length of the Red α 7029 is 660 base-pairs, its nucleosides Acid sequence is as shown in SEQ ID No.1,219 amino acid encodings of the gene code, its amino acid sequence SEQ ID No.5 institutes Show；The mrna length of the Red β 7029 is 873 base-pairs, and its nucleotide sequence is as shown in SEQ ID No.2, the gene 291 amino acid encodings are encoded, shown in its amino acid sequence SEQ ID No.6.

2. a kind of expression vector containing homologous recombination enzyme described in claim 1, it is characterised in that the carrier is with wide host Build and obtain based on replicon pBBR1, include pBBR1 replication orgins, kalamycin resistance gene (km^R), rhamnose induction Type promoter (P_Rha) and the homologous recombination enzyme Red β -7029/Red α -7029 that are reached by rhamnose inducible promoter control table；Life Entitled-the Km of pBBR1-Rha-BA 7029, its nucleotide sequence is as shown in SEQ ID No.3.

3. homologous recombination enzyme described in claim 1 is combined with from E.coli double-stranded DNA Exonucleolytic enzyme level albumen Red γ Application in Burkholderia In vivo recombination efficiency is improved.

4. application according to claim 3, it is characterised in that application process is：

(1) based on the replicon pBBR1 of wide host, structure includes pBBR1 replication orgins, kalamycin resistance gene (km^R), rhamnose inducible promoter (P_Rha) and reached by rhamnose inducible promoter control table recombinase Red γ- E.coli/Red β -7029/Red α's -7029 carries outside the homologous recombination enzymes of Red α/Red β 7029 and E.coli double-stranded DNA nucleic acid Enzyme cutting suppresses albumen Red γ recombinase expression vector, is named as-the Km of pBBR1-Rha-RedG-BA 7029, its nucleotides sequence Row are as shown in SEQ ID No.4；

(2) it is excellent to competent cell Optimization of preparation, the optimization of DNA inversion quantities, antibiotic selection concentration optimization, homologous arm lengths Change, it is determined that optimal condition of work is：Initial OD values be 0.1 the bacterium solutions of DSM 7029,30 DEG C, 950rpm cultivate 14 hours after, Add the 10mg ml of volume ratio 2%^-1Sandlwood sugar juice, 30 DEG C, 950rpm, 1.5h is induced, at the double distilled deionized water of normal temperature Manage competent cell and carry out electric conversion in normal temperature；

(3) under the condition of work of optimization, in (the Polyangium brachysporum strain of Burkholderia DSM 7029 DSM 7029) in carry out gene substitution, gene with frame delete and gene insertion, obtain mutant strain as described below：Using 50 ± The short homology arms of 10bp carry out a step knockout to a series of 100kb of the genomes of DSM 7029,200kb, 500kb fragments, obtain phase Answer a series of mutant strains；Pass through the measure to these mutant strain growth curves, it was demonstrated that large fragment is carried out to the genomes of DSM 7029 Knockout can significantly improve the growth rate and biomass of the mutant strain.

5. application of the homologous recombination enzyme described in claim 1 in compound glidopeptin is obtained.

6. application according to claim 5, it is characterised in that application process is：Utilize homologous recombination described in claim 1 Gene cluster of the enzyme to Burkholderia DSM 7029 (Polyangium brachysporum strain DSM 7029) genome Cluster 6A carry out the insertion of promoter and knock out deactivation maneuver, build the mutant strains of DSM 7029, are obtained by its tunning Compound glidopeptin is obtained, its concrete operation step and feature are as follows：

Promoter fragment of the both ends with 50bp homology arms is inserted into the genes of DSM 7029 first with Red α/Red β 7029 The P of DSM 7029 are built before cluster cluster 6A core gene_apraCluster 6A mutant strains, while utilize Red α/Red β 7029 are substituted with the gene cluster cluster 6A of resistant gene DSM 7029 of the both ends with 50bp homology arms nucleus The inactivation structure Δ cluster 6A mutant strains of DSM 7029, pass through the P of HPLC comparative analysis DSM 7029_apra cluster 6A With the Δ cluster 6A of DSM 7029 and wild type DSM 7029 fermentation crude extract, the P of DSM 7029 are determined_apra Cluster 6A can produce a kind of new compound, and this kind of Compound nomenclature is glidopeptin, and its charge-mass ratio is：m/z 676.2, [M+H]²⁺。

7. application of the homologous recombination enzyme described in claim 1 in compound rhizopeptin is obtained.

8. application according to claim 7, it is characterised in that application process is：It is homologous heavy using RedG-Red α β -7029 Group enzyme instead of the phosphopan tetheine sulfydryl second of the bacterial strains of P.rhizoxinica HKI 454 (DSM 19002) with resistant gene respectively Amido transferase gene (PPTase) and rhizomycin biological synthesis gene cluster (rhiz) construct the Δ PPTase of DSM 19002 and dashed forward Mutant and DSM19002 Δ rhiz mutant strains, tunning is analyzed by HPLC/MS and shown：In the wild types of DSM 19002 and A metabolite in tunning in DSM19002 Δ rhiz mutant strains be present：m/z 732.38,[M+H]⁺, and in DSM This metabolite disappears in 19002 Δ PPTase mutant strains, and it is probably Non-ribosomal peptide approach to prompt this metabolite Or the product of polyketide synthase approach, and in the mutant strain of rhizomycin synthetic gene cluster inactivation the product yield far above wild Type；Using Red α β -7029 homologous recombinations enzymes by being struck one by one to DSM 19002 PKS/NRPS biological synthesis gene clusters Except discovery：The compound disappears after cluster 1 on to the indigenous plasmids of DSM 19002 is knocked out；Further in the base After inserting constitutive promoter before cluster, it is found that the yield of the compound significantly improves, so that it is determined that the compound Synthetic gene cluster, and the yield of the compound is improved by promoter insertion, and it is named as rhizopeptin.