CN110129355A - The method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination - Google Patents

The method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination Download PDF

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CN110129355A
CN110129355A CN201910359121.8A CN201910359121A CN110129355A CN 110129355 A CN110129355 A CN 110129355A CN 201910359121 A CN201910359121 A CN 201910359121A CN 110129355 A CN110129355 A CN 110129355A
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ura4
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saccharopolyspora strain
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pogona
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夏立秋
穰杰
何昊城
丁学知
孙运军
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Hunan Normal University
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Abstract

The method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination, comprising the following steps: (1) must find ura4 as negative selection marker gene in saccharopolyspora strain genome；(2) sequence that design separately designs that one section of size is 1 kb at the both ends of ura4 gene is used to carry out the homology arm of ura4 gene replacement；(3) ura4 gene upstream and downstream homology arm is merged using fusion DNA vaccine with A Bola resistant gene, and is attached with plasmid pOJ260, obtain recombinant plasmid pOJ260-ura4；(4) protoplast containing recombinant plasmid pOJ260-ura4 is transferred in palpus saccharopolyspora strain, carry out the knockout of ura4 gene, ura4 deletion mycopremna S.pogona- Δ ura4 is obtained, carries out the blocking of palpus saccharopolyspora strain gene cluster to the ura4 deletion mycopremna S.pogona- Δ ura4 by linear fragment homologous recombination technique.The present invention can obtain the positive monoclonal blocked with target gene cluster without carrier construction, introducing external source recombinase in a short time, improve the efficiency that the non-target gene cluster of palpus saccharopolyspora strain blocks.

Description

The method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination
Technical field
The present invention relates to a kind of methods that palpus saccharopolyspora strain carries out gene cluster blocking, and in particular to one kind is based on linear fragment Homologous recombination carries out the method that palpus saccharopolyspora strain gene cluster blocks.
Background technique
Butenyl spinosyn is the macrolides compound that the one kind generated by palpus saccharopolyspora strain has insecticidal activity, And there is insecticidal activity more stronger than pleocidin and insecticidal spectrum, the worldwide quarantine pest that such as pleocidin is difficult to control There is good biological control effect with important agricultural insect oriental tobacco budworm, it has also become current new bio agriculture most with prospects One of medicine.But the ability of wild type palpus saccharopolyspora strain synthesis butenyl spinosyn is weaker and fermentation period is long, restricts fourth The large-scale industrial production of alkenyl pleocidin and application.Therefore, how to shorten fermentation period and improve cyclobutenyl and kill more The yield of rhzomorph becomes current hot issue urgently to be solved.
The biosynthesis of butenyl spinosyn is that (Polyketide synthase, polyketone close using typical I type PKS Enzyme) approach completes, carries out condensation reaction repeatedly by catalytic precursor substance, form polyketide, then carry out glycosylation and Methylation modification forms final product.However, the progress of the process may be by the competition of other secondary metabolite gene clusters Property inhibit, it is such as competitive to utilize precursor substance.Must saccharopolyspora strain genome sequencing the results show that the strain gene group exist remove Other 27 secondary metabolite gene clusters outside butenyl spinosyn gene cluster.The expression of these gene clusters is likely to be such that The supply of butenyl spinosyn biosynthesis precursor is reduced, final to influence butenyl spinosyn biosynthesis.Therefore, pass through resistance Non-target gene cluster break to reduce the utilization to synthesis precursor, is expected to there are efficacious prescriptions as raising butenyl spinosyn yield Method.As Meng et al. in Avid kyowamycin by being blocked to III type gene cluster rpp, promote avermectin yield by 1, 024 mg/L is increased to 1,262 mg/L(Meng L, Xiong Z, Chu J, Wang Y. Enhanced production of avermectin by deletion of type III PKSs biosynthetic cluster rpp in Streptomyces avermitilis. Lett Appl Microbiol. 2016 Nov;63(5):384-390. doi: 10.1111/lam.12635.).
Currently, built erect a variety of genetic modification systems to carry out microbial genome editor, such as CRISPR/cas9 gene Group editing technique (Aamir Mir, Alireza Edraki, Jooyoung Lee, and Erik J. Sontheimer. Type II-C CRISPR-Cas9 Biology, Mechanism, and Application. ACS Chem Biol. 2017, doi:10.1021/acschembio.7b00855.), Red/ET homologous recombination technique (Hailong Wang, Zhen Li, Ruonan Jia, Yu Hou, Jia Yin, Xiaoying Bian, Aiying Li, Rolf Müller, A Francis Stewart, Jun Fu, Youming Zhang. RecET direct cloning and Red αβ recombineering of biosynthetic gene clusters, large operons or single genes For heterologous expression. Nat Protoc. 2016,11 (7): 1175-1190.), the site Cre/loxP Specific recombinant technique (Zhengqiang Liu, Yali Xie, Xu Zhang, Xiaofeng Hu, Yusheng Li, Xuezhi Ding, Liqiu Xia, Shengbiao Hu. Efficient Construction of Large Genomic Deletion in Agrobacterium tumefaciens by Combination of Cre/loxP System and Triple Recombineering. Curr Microbiol. 2016,72 (4): 465-472.) and I-SceI inscribe enzyme system Unite (Zhongqiu Chen, Wen Ling, Guangdong Shang. Recombineering and I-SceI- mediated Pseudomonas putida KT2440 scarless gene deletion. FEMS Microbiology Letters. 2016,363 (21): fnw231.) etc., it is suitable for microbial genome large fragment knockout.But these are lost It passes operating system to require through plasmid-mediated or introducing external source recombinase, some also need to carry out the ability of plasmid conversion twice Genome editing is completed, since palpus saccharopolyspora strain exogenous plasmid conversion is extremely difficult, to limit above-mentioned genetic modification Quick application of the system in palpus saccharopolyspora strain.
The homologous recombination technique that existing research shows that linear fragment mediates is equally applicable to microbial genome editor, such as Mayumi et al. by linear fragment recombinate 657.3 kb sequence of successful knockout Yeast genome (Mayumi Sasaki, Hiromichi Kumagai, Kaoru Takegawa, Hideki Tohda. Characterization of genome- reduced fission yeast strains. Nucleic Acids Research. 2013, 41(10):5382- 5399.).In addition to yeast, the recombinant technique is in other microorganisms such as Bacillus subtillis also Successful utilization.
Currently, related palpus saccharopolyspora strain genome editing predominantly stays in single-gene level, gene cluster large fragment is hindered Disconnected research yet there are no report, and exogenous plasmid conversion is also more difficult.
Summary of the invention
The technical problem to be solved by the present invention is to, overcome drawbacks described above of the existing technology, provide it is a kind of can be to base Because cluster carry out large fragment blocking, and without carrier construction, introduce external source recombinase based on linear fragment homologous recombination carry out palpus The method that saccharopolyspora strain gene cluster blocks.
The technical solution adopted by the present invention to solve the technical problems is as follows: one kind is carried out based on linear fragment homologous recombination The method that palpus saccharopolyspora strain gene cluster blocks, comprising the following steps:
(1) ura4 must found as negative selection marker gene in saccharopolyspora strain genome；
(2) sequence that design separately designs that one section of size is 1 kb at the both ends of ura4 gene is used to carry out ura4 gene replacement Homology arm；
(3) ura4 gene upstream and downstream homology arm is merged with A Bola resistant gene using fusion DNA vaccine, and and plasmid POJ260 is attached, and obtains recombinant plasmid pOJ260-ura4；
(4) protoplast containing recombinant plasmid pOJ260-ura4 is transferred in palpus saccharopolyspora strain, carries out striking for ura4 gene It removes, ura4 deletion mycopremna S.pogona- Δ ura4 is obtained, by linear fragment homologous recombination technique to the ura4 deletion mycopremna S.pogona- Δ ura4 carries out the blocking of palpus saccharopolyspora strain gene cluster.
Further, ura4 gene order described in step (1) is as shown in sequence table SEQ ID № 1.
Further, for replacing the upstream homology arm sequence such as sequence table SEQ ID № of ura4 gene described in step (2) Shown in 2；The downstream homology arm sequence for replacing ura4 gene is as shown in sequence table SEQ ID № 3.
Further, replace ura4 gene using apramycin resistance gene described in step (3), making must saccharopolyspora strain tool There is apramycin resistance, in the subsequent saccharopolyspora strain progress transformant screening to palpus, adds apramycin.
Further, step (4) obtains ura4 deletion mycopremna S.pogona- Δ ura4 and carries out as palpus saccharopolyspora strain gene cluster The type strain that large fragment blocks.
Further, step (4) the linear fragment homologous recombination technique that passes through is to the ura4 deletion mycopremna S.pogona- Δ ura4 carries out the blocking of palpus No. 14 gene clusters of saccharopolyspora strain, comprising the following steps:
(a) blacked-out areas downstream is waited in No. 14 gene clusters, genome is upper to be set to 2,627,531~2,631,482, designs two pairs Primers F c14up/Rc14up and Fc14down/Rc14down carry out the amplification of upstream and downstream homology arm, for linear fragment to be inserted into In genome；
(b) in No. 14 gene cluster region upstreams to be knocked out, genome is upper to be set to 2,609,020~2,609,519, and design is a pair of Primers F ds/Rds carries out direct sequence amplification, knocks out outside the external source ura4 gene ring for that will introduce；
(c) pair of primers Fura4/Rura4 progress ura4 gene magnification must be being designed in saccharopolyspora strain genome, as negative selection Marker gene carries out transformant screening；
(d) using plasmid pOJ260-cm-Perm* as template, design pair of primers Fperm/Rperm is used for erythromycin strong promoter (Perm*) it expands, for starting ura4 gene expression；
(e) genetic fragment that step (a) to step (d) obtains is merged using fusion DNA vaccine, acquisition can be used for No. 14 genes The linear recombinant fragment UAc14-Perm*-ura4-DS-DAc14 that cluster blocks；
(f) the linear recombinant fragment of protoplast transformation, which enters in S.pogona- Δ ura4, is recombinated, is screened, and No. 14 bases are obtained The engineered strain S. pogona- Δ ura4- Δ c14 blocked by cluster.
Further, the engineered strain S. pogona- Δ that resulting palpus No. 14 gene clusters of saccharopolyspora strain of step (f) block The blacked-out areas of ura4- Δ c14 is 20 kb sequences.
The present invention, must the more spores of sugar using double crossing over homologous recombination on the basis of determining negative selection marker gene ura4 Ura4 gene knockout in bacterium genome obtains the mutant strain S. pogona- Δ ura4 for being free of ura4 gene, that is, completes to carry out The building for required " chassis " cell that gene cluster large fragment blocks；Physiology, biochemical studies show ura4 gene knockout not Influence the growth, hypha form change and butenyl spinosyn biosynthesis of starting strain；Treat No. 14 genes of blocking The design that cluster carries out linear fragment (includes two homology arms, a negative selection marker gene ure4 and an orientation repeat sequence Column), it assembles (fusion DNA vaccine), and be transferred in mutant strain S. pogona- Δ ura4 protoplast, uses 5-FOA (5- fluorine whey Acid) negative selection is carried out, the engineered strain S. pogona- Δ ura4- Δ 14 that gene cluster cluster 14 is blocked is obtained, and PCR reflects The result shows that, successfully the nucleus of cluster 14 (about 20 kb) is blocked calmly.
Invention, can also be quickly the utility model has the advantages that in the use that must not only avoid plasmid in saccharopolyspora strain genome editor The mutant strain blocked to specific gene cluster is obtained, is the powerful for carrying out palpus saccharopolyspora strain genome editor.Moreover, of the invention Pass through engineered strainS. pogona-Δura4-The acquisition of Δ 14, it was demonstrated that by blocking competitive gene cluster to shared precursor The utilization of matter can effectively improve butenyl spinosyn yield.
Detailed description of the invention
Fig. 1 is the distribution of the ura4 and upp that can be used as negative selection marker gene of the present invention in the genome；
Fig. 2 is that the recombinant plasmid pOJ260-ura4 of the present invention constructs flow chart；
Fig. 3 is that the mutant strain S. pogona- Δ ura4 of the present invention constructs flow chart；
Fig. 4 is the mutant strain bacterium S. pogona- Δ ura4 PCR proof diagram of the present invention；
Fig. 5 is that the mutant strain bacterium S. pogona- Δ ura4 growth curve of the present invention measures；
Fig. 6 is that the 3rd day, the 5th day and the 7th day hypha form scanning electron microscope of mutant strain bacterium S. pogona- Δ ura4 of the present invention is seen It examines；
Fig. 7 is that mutant strain bacterium S. pogona- Δ ura4 (A) and original strain S. pogona (B) cyclobutenyl of the present invention kills more Rhzomorph volume analysis；
Fig. 8 is the flow chart for the method that the linear fragment of the present invention mediates palpus saccharopolyspora strain genome large fragment to knock out；
Fig. 9 is palpus No. 14 gene cluster genomic organizations of saccharopolyspora strain of the present invention；
Figure 10 is that the linear recombinant fragment (A) of the present invention and 14 (B) PCR of engineered strain S. pogona- Δ ura4- Δ are verified Figure；
Figure 11 is the engineered strain S. pogona- Δ ura4- Δ 14 (A) and original strain S. pogona (B) butylene of the present invention Quito bacteriocidin volume analysis.
Specific embodiment
Below with reference to embodiment and attached drawing, the invention will be further described.
Chemical reagent used in the embodiment of the present invention is obtained by routine business approach unless otherwise specified.
Embodiment 1
(1) specific culture medium prescription and condition of culture
(1) S. pogona seed activation culture medium C SM (Complete synthetic medium): TrypticSoyBroth 4.5g/L, Glucose 0.9g/L, Yeast extract 0.3g/L, MgSO4.7H2O 0.22g/L.It is inoculated with monoclonal or 2% bacterium Kind preservative fluid is added in the 150 mL shaking flasks equipped with 20 mLCSM, and 30 °C, 280r/min cultivates 48 h;
(2) S. pogona synthesizes fermentation medium (Synthetic fermentation medium, SFM): Glucose 20.0 g/L, Yeast extract 4.0 g/L, Tryptone 4.0 g/L, KNO31.0 g/L, K2HPO4•3H2O 0.5 G/L, MgSO4•7H2O 0.5 g/L, FeSO4•7H2O 0.01 g/L.The actication of culture liquid of inoculation 2%, which is added, is equipped with 50 mL SFM 300mL shaking flask in, 30 °C, 280r/min cultivate 12 d.
(3) protoplast transformation uses R5 culture medium: 103 g/L of sucrose, 10 g/L of glucose, casamino acid 0.1 g/L, Yeast extract 5 g/L, K2SO40.25 g/L, MgCl2•6H2O 10.12g/L, TES 5.73 g/L, it is micro- 2 ml (ZnCl of secondary element solution240 mg/L, FeCl3•6H2O 200 mg/L, CuCl2•2H2O 10 mg/L, MnCl2• 4H2O 10 mg/L, Na2B4O7•10H2O 10 mg/L, (NH4)6Mo7O4•4H2O 10mg/L), agar powder 15-20 g/L, The KH of filtration sterilization is separately added into before use in the medium2PO3(0.5%) 2.5 mL/L, L-PROLINE (20%) 3.75 mL/L and CaCl2•2H2O (5 M)1 mL/L。
(4) E.coli DH5 α fluid nutrient medium LB (Luria-Bertani): Tryptone 1 g/L, Yeast 1 g/L of extract 0.5 g/L and NaCl.The agar powder that 15-20 g/L is added in aforesaid liquid can be configured to the training of LB solid Support base.
(2) must in saccharopolyspora strain genome negative selection marker gene analysis
In reported document, (uracil turns phosphoric acid by big more options ura4 (orotidine -5- phosphate decarboxylase gene) and upp Ribosyl enzyme) these two types of gene is as negative selection marker gene.Wherein orotidine -5- phosphate decarboxylase is in 5-FOA (5- fluorine whey Acid) in the presence of can be translated into 5-FU (5 FU 5 fluorouracil), and uracil transphosphoribosylase can convert 5-FU to 5-fluoro-UMP (5-FUD acid), the final activity for inhibiting thymidylate synthetase cause cell death.The two negative selections The screening conditions of marker gene are 5-FOA and 5-FU respectively.Must saccharopolyspora strain genome sequencing the result shows that, the bacterial strain exist 1 ura4 gene and 2 upp genes, at genomic locations (referring to Fig. 1).It therefore, is to simplify operation, our final choices The negative selection gene that ura4 gene is invented as this.
(3) ura4 gene knockout plasmid construction
Using S. pogona genome as template, two couples of primers Fs ura4up/Rura4up and Fura4down/Rura4down are designed Homology arm of each 1 kb or so sequence as double crossing over is expanded in the upstream and downstream of ura4 gene respectively, primer sequence is shown in Table 1 institute Show, wherein primers F ura4up and Rura4down is respectively provided with Hind III and EcoRI restriction enzyme site, will be used for and carrier pOJ260 It is attached.The PCR reaction system of two sections of homology arms amplification are as follows: H216.5 μ L, 2X PrimeSTAR GC Buffer 25 of O 2 0.5 μ L of μ L, PrimeSTAR of 4 μ L of μ L, dNTPs, primer each 2 μ L, template S. pogona genomic DNA.PCR reaction Program setting are as follows: 95 DEG C of initial denaturation 5min then press 30s and 72 DEG C of 95 DEG C of denaturation 30s, 56 DEG C of annealing extension 90s, circulation 30 Secondary, finally 72 DEG C of maintenance 10min, 20 DEG C of terminations are reacted again.
Using plasmid pOJ260 as template, design pair of primers Fapr/Rapr draws for expanding apramycin resistance gene Object sequence is shown in Table 1.5 ' the ends of above-mentioned primer pair Fapr/Rapr respectively can be homologous with the upstream and downstream of ura4 gene with 35bp The sequence of arm overlapping, can be used for carrying out three segment compositions.PCR reaction system and response procedures and ura4 gene upstream and downstream are homologous Arm setting is consistent.
Above-mentioned 3 kinds of PCR products are purified, are recycled, and using the recovery product of acquisition as template, Fura4up and Rura4down is primer, using Fusion PCR method, by ura4 gene upstream and downstream homology arm and apramycin resistance base Because apr carries out seamless spliced, three segment fusion product UAura4-apr-DAura4 (Fusion fragment) of acquisition.Then Hind III and EcoRI double digestion (double digestion system: ddH are carried out respectively to Fusion fragment and pOJ262O 10 μ L, 10 5 μ L, Fusion fragment/pOJ260 of × M buffer, 30 III 55 μ L of μ L, EcoRI of μ L, Hind), product recycling After purification, fusion segment is carried out under the action of T4DNA ligase, and (enzyme disjunctor system: T4 DNA is connect with carrier pOJ260 POJ260: Fusion fragment of ligase buffer 1:T4 DNA ligase:Linear=1:1:1:7).Finally Connection product heat is transferred in E.coli DH5 α competent cell, and is converted on LB resistant panel (apramycin) Son screening and verifying obtain ura4 and knock out plasmid pOJ260-ura4, construction of recombinant plasmid process (referring to fig. 2).
(4) building of mutant strain S. pogona- Δ ura4
External source recombinant plasmid pOJ260-ura4 is transferred in S. pogona using the mode of protoplast transformation, the above plasm Body preparation can refer to Luo Yushuan, Ding Xuezhi, Xia Liqiu, Wang Hailong, yellow Fan, Tang Ying " bioengineering journal " with method for transformation Delivered on (the 3rd the 360-367 pages of phase of volume 25 in 2009) " pleocidin produces the preparation condition and its again of bacterium protoplast The progress that raw bacterial strain physiological property " article is recorded.It after obtaining transformant, places it in TSB fluid nutrient medium, 30 DEG C of shaking table trainings Support 48 h.It collects 1 mL bacterium solution and extracts genome as template, carry out apramycin resistance using primer pair Fapr/Rapr Gene magnification, and using wild type S. pogona genome as negative control, recombinant plasmid pOJ260-ura4 be positive control (by Belong to suicide type plasmid in pOJ260, cannot be replicated in S. pogona, is caused after passing on n times, which can lose, from Interference is generated without identifying transformant).PCR is identified into correct transformant (mutant strain), is named as S. pogona- Δura4.As a result (referring to Fig. 3 and Fig. 4), S. pogona- Δ ura4 has when carrying out PCR amplification with primer pair Fapr/Rapr The band of about 1.5 kb occurs, and wild type S. pogona occurs without corresponding band, illustrates that apr gene successfully replaces Ura4 gene is so far constructed for host strain needed for carrying out seamless knockout and is completed.
(5) mutant strain S. pogona- Δ ura4 phenotypic analysis
(1) influence of the ura4 gene knockout to strain growth
Activated mutant strain S. pogona- Δ ura4 and original strain equivalent are transferred in synthesis fermentation medium respectively, (measured value between 0.2 ~ 08, to guarantee the accuracy of measurement result, otherwise every 12 h sampling carries out the measurement of thallus OD600 value Sample is diluted), the sampling period is 12 d.Experimental result (referring to Fig. 5), mutant strain S. pogona- Δ ura4 and original Beginning bacterial strain S., pogona was compared, and growth curve is without significant change.
(2) influence of the ura4 gene knockout to bacterial strain hypha form
Activated mutant strain S. pogona- Δ ura4 and original strain equivalent are transferred in synthesis fermentation medium respectively, And the 3rd day, the 5th day and the 7th day bacterium solution is taken to be scanned Electronic Speculum observation.Sample is fixed through the glutaraldehyde of 2.5 % and ethyl alcohol After dehydration, using cold field emission scanning electron microscope SU8010 carry out mutant strain S. pogona- Δ ura4 with it is original The observation of bacterial strain mycelium morphology.Scanning electron microscopic observation is as the result is shown (referring to Fig. 6), mutant strain S. pogona- Δ ura4 mycelia Volume morphing is Filamentous with the 5th day presentation branch on day 3, cultivates to after the 7th day, mycelium morphology gradually becomes mycelia breaking state And few branch, mycelium change procedure are consistent with original strain S. pogona.
(3) influence of the ura4 gene knockout to butenyl spinosyn biosynthesis
Activated mutation S. pogona- Δ ura4 and original strain equivalent are transferred in synthesis fermentation medium respectively, take training It supports to the 10th day fermentation liquid and carries out the detection of butenyl spinosyn yield.Sample handling processes may participate in document (Luo Lingen, poplar Swallow, Wei Hui, the stalks of rice, wheat, etc. are outstanding, and Tang Qiong, Hu Shengbiao, Sun Yunjun, minor is complete, Ding Xuezhi, and Xia Liqiu was " bioengineering journal " (2016 years the The 259-263 pages of the phase of volume 32 the 2nd) on deliver " must saccharopolyspora strain Saccharopolyspora pogona ribosome engineering The progress that the influence that transformation synthesizes butenyl spinosyn " article is recorded.HPLC1290 testing result (referring to Fig. 7), mutation Bacterial strain S. pogona- Δ ura4 is respectively with original strain S. pogona butenyl spinosyn peak area, statistically without Notable difference.
Above-mentioned phenotypic analysis the results show that do not sterilize strain growth, development and cyclobutenyl after ura4 gene knockout more Plain biosynthesis has an adverse effect, and illustrates correct using ura4 gene as palpus saccharopolyspora strain negative selection marker gene selection.
(6) linear fragment homologous recombination technique is used for the blocking of target gene cluster cluster 14
There are 28 secondary metabolite gene clusters in palpus saccharopolyspora strain genome, we randomly choose No. 14 gene clusters, non-core Sugared body polypeptide gene cluster (referring to Fig. 9), the verifying of above-mentioned large fragment knockout technique is carried out as target.
(1) linear recombinant fragment UAc14-Perm*-ura4-DS-DAc14 external structure
Using S. pogona genome as template, four couples of primers Fs ura4/Rura4, Fc14up/Rc14up, Fc14down/ are designed Rc14down and Fds/Rds carries out ura4 gene, upstream and downstream homology arm and orientation repetitive sequence amplification, is respectively used to carry out weight Group bacterial strain negative selection, linear recombinant fragment are integrated into genome and negative selection marker gene (ura4) is deleted, and primer sequence is such as Shown in table 1, wherein the 5 ' of primers F c14down, Fsd and Rsd respectively are used to carry out the base sequence of segment composition added with 35 bp Column.PCR reaction system and response procedures are consistent with above-mentioned setting.
The bacterial strain uses therefor of the present invention of table 1, plasmid and primer
Using plasmid pOJ260-cm-Perm* as template, design pair of primers Fperm/Rperm is for expanding erythromycin strong promoter (Perm*), primer sequence is as shown in table 1, and wherein the 5 ' of primer Rprem are used to carry out the base of segment composition added with 35 bp Sequence.PCR reaction system and response procedures are consistent with above-mentioned setting.
Finally, above-mentioned 5 kinds of PCR products are carried out recovery purifying, and using the recovery product of acquisition as template, Fc14up with Rc14down is primer, using Fusion PCR method, above-mentioned 5 kinds of segments is carried out " suture ", obtain five fusion products (UAc14-Perm*-ura4-DS-DAc14), construction method (referring to Fig. 8).
(2) building of engineered strain S. pogona- Δ ure4- Δ 14
Linear recombinant fragment UAc14-Perm*-ura4-DS-DAc14 is transferred to S. using the mode of protoplast transformation In pogona- Δ ura4 and recombinate；100 μ l bacterium solutions are taken to be coated on MM (basal medium) plate containing 5-FOA enterprising Row negative selection, 30 DEG C of constant temperature incubation carton upside down cultures are until white transformant occurs；Picking monoclonal is lined containing 5-FOA's It is isolated and purified on MM plate, after repeating 2-3 times, obtains final transformant.
Picking part lawn is in TSB fluid nutrient medium on MM plate, 30 DEG C of 48 h of shaking table culture, collects 1 mL bacterium solution And it extracts genome and carries out PCR verifying using primer pair Fsd/Rc14down as template, and with wild type S. pogona base Because group is negative control.PCR identifies correct transformant (engineered strain), is named as S. pogona- Δ ura4- Δ c14.Knot Fruit shows that S. pogona- Δ ura4- Δ c14 will appear about 1.8 kb when being verified with primer pair Fsd/Rc14down Band, and original strain S. pogona without band occur (referring to Fig. 9), illustrate successfully to the gene cluster target area into Row blocks.
(7) engineered strain S. pogona- Δ ura4- Δ c14 butenyl spinosyn volume analysis
Respectively by OD600 values such as activated engineered strain S. pogona- Δ ura4- Δ c14 and original strain S. pogona It is transferred in synthesis fermentation medium, the fermentation liquid of culture to the 10th day is taken to carry out the detection of butenyl spinosyn yield. HPLC1290 testing result is as shown in Figure 10, engineered strain S. pogona- Δ ura4- Δ c14 and original strain S. pogona It compares, in addition to the chromatographic peak of 5.3min, apparent chromatographic peak occurs in 8 min, which is that cyclobutenyl is sterilized through Mass Spectrometric Identification more Plain derivative spinosyn α a, therefore, engineered strain S. pogona- Δ ura4- Δ c14 butenyl spinosyn total peak area Respectively 1904.7, and original strain total peak area is 342.1 mAU*min, after final gene cluster cluster14 is blocked, so that Butenyl spinosyn output increased 5.57 times.
Sequence table
<110>Hunan Normal University
<120>method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 849
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 1
atgactgatc cgctgcgggc gggcttcggc cagcggctgg cggagtcggt gcgggcgcgc 60
ggtgcgctgt gcgtcggcat cgacccgcac ccgtcgctgc tgcacgcgtg ggatctcgac 120
gagaccccgc agtcgctgga gcggttcgcg atgaccgcgg tggaggcgct ggccggcgag 180
gtcgcggtgc tcaagccgca gtcggcgttc ttcgaggtgt acggctcggc cggaatcgcc 240
gtgctggaga agacgatccg ggaggcgcgg caggccggtg cgctggtgct gctggacatc 300
aagcgcggcg acatcggctc cacgatggcg gcgtacgcga tggcctacct ggacgagcac 360
gcgccgctgg cggcggatgc gatcacggtg tcgccgtacc tcggctacgg ctcgctcgcc 420
ccggcgatcg gcatcgccga gcagacgggc cgcggggtgt tcgtgctggc gcgcacctcc 480
aacccggagg gcgcgggcct ccagcggtcg atccacggca gcggcgactc ggtcgcccag 540
tacatcgtgg actcggccgc ggagaccaat gcggacgcgg agccgatggg gcacgtcgga 600
gtggtggcgg gagccaccat cggcgccgac gagctggact tctcccgcct caacggcccg 660
atcctggcac ccggcctggg tgcgcagggt gcgacggtgc agagcctgcg cgcgctgttc 720
ggcgcggcgc tgccgaacgt cctaccggcg acggcccgcg acgtcctccg gcacggcccg 780
acggtcaacg gcctccgcgc cgcggcccac aaggtccgcg acgaggtgag cgacctcctc 840
cgagactga 849
<210> 2
<211> 1274
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 2
tggtggacaa cttcctcgac gacgcgatcg agatcgacgt ggacgcgctc tgcgacggca 60
ccgacatcta cctcggtggc gtgatggaac acatcgagga ggccggtatc cactccggcg 120
actcggcgtg cgcgctgccg ccgatcacgt tggggcgtca ggacatcgag caggtgcgaa 180
agtccaccga ggccatcgcg cgcggcatcg gcgtgcacgg cctgctgaac gtgcagtacg 240
cgctcaagga cgacgtgctg tacgtgctgg aggccaaccc gcgcgcctcg cggaccgtgc 300
cgttcgtgtc caaggcgacc gccgtgccgc tggccaaggc ggcggcccgg atcatgctcg 360
gcgccaagat cgcggacctg cgcgcggagg gcctgctgcc gccggagggc gacggcgcgg 420
aactgccgat cgacgcgccg gtcgcggtga aggaagccgt gctgccgttc caccggttcc 480
gcacccgcga gggcgtcggc gtggactcgc tgctcgggcc ggagatgaag tccaccggcg 540
aggtgatggg catcgacacc tccttcgggc aggccttcgc caagtcgcag gccggcgcct 600
acggttcgct gccgaccggc ggccgggtgt tcgtgtcggt ggccaacaag gacaagcggt 660
ccctggtgtt cccggtcaag cggctggcgg acctcggctt cgagatcctg gccaccagcg 720
gcaccgcgga ggtgctgcgc cgcaacggaa tcccgtccac ggtggtgcgc aagcacatcg 780
agaagaacgg cgcggagcgc gacatcgtcg agctgatcaa ggccggcgag gtcgacatgg 840
tgatcaacac cccgtacggc aacccgggcc cgcgcgtgga cggctacgag atccgcaccg 900
ccgcggtatc ccgcgacatc ccgtgcatca ccacggtgca gggcgcggcg gcggccgtgc 960
agggcatcga ggccgccatc caaggcaaca tcggggtccg gcccctgcag gcgttgcagg 1020
cggcccttcg gcagggcggt gagcagcgat gactgatccg ctgcgggcgg gcttcggcca 1080
gcggctggcg gagtcggtgc gggcgcgcgg tgcgctgtgc gtcggcatcg acccgcaccc 1140
gtcgctgctg cacgcgtggg atctcgacga gaccccgcag tcgctggagc ggttcgcgat 1200
gaccgcggtg gaggcgctgg ccggcgaggt cgcggtgctc aagccgcagt cggcgttctt 1260
cgaggtgtac ggct 1274
<210> 3
<211> 1172
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 3
ggaaacggct atctggtacg aagttcgcat gcatcccgca acgaacggcg ccctcgcgca 60
gaagaaaccg ccgcgcgtgc tgcgctggat catcgttctc gtgctgctct ggctggtcac 120
cggtggctat ttcctggttt ccaccgaggt caccatctgg tcccggacct tcggctcggg 180
cgaggccacg gccgacccag tcgcacacgt ggagctggag cgggggcagc cgttcggcct 240
ggccgcgcaa cgggatgagt acgtgaagtg ccaggtgatc ccgcaggccg gtgagccgcg 300
tgagttcatt cccgaccgcc cggccaacag cctgcgcagc tcgcggctgc ccgggccgga 360
acccgcgtgg ttctccggcc cggccgaggc ccgctgcacc gggcccacca cggtgctcct 420
gccggagcgc tacgacacga ccgggctgaa cgtcctgctg gggctgattg ctgcggccag 480
cctcatgatc gtggtcgttt ttgtacaatt cgcgcgtcgt gcccgtttca acaataggtt 540
gtacgcccgc gactgatttg ctcctgggaa cggatctccg aacggcatga ggcgccgtgg 600
gcggtcagat agtgtcgatc caacggttcg gaagctaagg ggacagacgg tgagcagcgc 660
gtctgagcgc atccaggaga tgatgcgcaa gttcgaggag caggcgcaga aggcgtcgga 720
actgcagtcc gcgatgcagg gcatgcaggc cacggcgagc agcccggacc ggtcggtgac 780
ggtgacggtc gcgccgtccg gggcggtgct ggacctgcgt ctggcgccga acgcggtgcg 840
ccagtcggcc aacgacctgc agcagcagat catggcgacc atccgccaag caacggcaag 900
cgcggcggag cagctcaaca acacggtggc gccgatcctg ggcgaccagt tcgacaagtt 960
ccaggaagcg ttcaacgtcg aaggcatggc gatcaagccg accgccggag acgaaacgcc 1020
cgcaccggag agccccgcac cgccgcccgc cgcgcaccgc gcgccgccgg agagtccggc 1080
aacgccgcgg cggccgcagc aggcgagcgc cgattacgac ggtgacgact tctcttcggg 1140
ctcgttcctg cgctgaggga agttgtcttg ac 1172
Sequence table
<110>Hunan Normal University
<120>method that palpus saccharopolyspora strain gene cluster blocks is carried out based on linear fragment homologous recombination
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 849
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 1
atgactgatc cgctgcgggc gggcttcggc cagcggctgg cggagtcggt gcgggcgcgc 60
ggtgcgctgt gcgtcggcat cgacccgcac ccgtcgctgc tgcacgcgtg ggatctcgac 120
gagaccccgc agtcgctgga gcggttcgcg atgaccgcgg tggaggcgct ggccggcgag 180
gtcgcggtgc tcaagccgca gtcggcgttc ttcgaggtgt acggctcggc cggaatcgcc 240
gtgctggaga agacgatccg ggaggcgcgg caggccggtg cgctggtgct gctggacatc 300
aagcgcggcg acatcggctc cacgatggcg gcgtacgcga tggcctacct ggacgagcac 360
gcgccgctgg cggcggatgc gatcacggtg tcgccgtacc tcggctacgg ctcgctcgcc 420
ccggcgatcg gcatcgccga gcagacgggc cgcggggtgt tcgtgctggc gcgcacctcc 480
aacccggagg gcgcgggcct ccagcggtcg atccacggca gcggcgactc ggtcgcccag 540
tacatcgtgg actcggccgc ggagaccaat gcggacgcgg agccgatggg gcacgtcgga 600
gtggtggcgg gagccaccat cggcgccgac gagctggact tctcccgcct caacggcccg 660
atcctggcac ccggcctggg tgcgcagggt gcgacggtgc agagcctgcg cgcgctgttc 720
ggcgcggcgc tgccgaacgt cctaccggcg acggcccgcg acgtcctccg gcacggcccg 780
acggtcaacg gcctccgcgc cgcggcccac aaggtccgcg acgaggtgag cgacctcctc 840
cgagactga 849
<210> 2
<211> 1274
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 2
tggtggacaa cttcctcgac gacgcgatcg agatcgacgt ggacgcgctc tgcgacggca 60
ccgacatcta cctcggtggc gtgatggaac acatcgagga ggccggtatc cactccggcg 120
actcggcgtg cgcgctgccg ccgatcacgt tggggcgtca ggacatcgag caggtgcgaa 180
agtccaccga ggccatcgcg cgcggcatcg gcgtgcacgg cctgctgaac gtgcagtacg 240
cgctcaagga cgacgtgctg tacgtgctgg aggccaaccc gcgcgcctcg cggaccgtgc 300
cgttcgtgtc caaggcgacc gccgtgccgc tggccaaggc ggcggcccgg atcatgctcg 360
gcgccaagat cgcggacctg cgcgcggagg gcctgctgcc gccggagggc gacggcgcgg 420
aactgccgat cgacgcgccg gtcgcggtga aggaagccgt gctgccgttc caccggttcc 480
gcacccgcga gggcgtcggc gtggactcgc tgctcgggcc ggagatgaag tccaccggcg 540
aggtgatggg catcgacacc tccttcgggc aggccttcgc caagtcgcag gccggcgcct 600
acggttcgct gccgaccggc ggccgggtgt tcgtgtcggt ggccaacaag gacaagcggt 660
ccctggtgtt cccggtcaag cggctggcgg acctcggctt cgagatcctg gccaccagcg 720
gcaccgcgga ggtgctgcgc cgcaacggaa tcccgtccac ggtggtgcgc aagcacatcg 780
agaagaacgg cgcggagcgc gacatcgtcg agctgatcaa ggccggcgag gtcgacatgg 840
tgatcaacac cccgtacggc aacccgggcc cgcgcgtgga cggctacgag atccgcaccg 900
ccgcggtatc ccgcgacatc ccgtgcatca ccacggtgca gggcgcggcg gcggccgtgc 960
agggcatcga ggccgccatc caaggcaaca tcggggtccg gcccctgcag gcgttgcagg 1020
cggcccttcg gcagggcggt gagcagcgat gactgatccg ctgcgggcgg gcttcggcca 1080
gcggctggcg gagtcggtgc gggcgcgcgg tgcgctgtgc gtcggcatcg acccgcaccc 1140
gtcgctgctg cacgcgtggg atctcgacga gaccccgcag tcgctggagc ggttcgcgat 1200
gaccgcggtg gaggcgctgg ccggcgaggt cgcggtgctc aagccgcagt cggcgttctt 1260
cgaggtgtac ggct 1274
<210> 3
<211> 1172
<212> DNA
It<213>must saccharopolyspora strain (Saccharopolyspora pogona)
<400> 3
ggaaacggct atctggtacg aagttcgcat gcatcccgca acgaacggcg ccctcgcgca 60
gaagaaaccg ccgcgcgtgc tgcgctggat catcgttctc gtgctgctct ggctggtcac 120
cggtggctat ttcctggttt ccaccgaggt caccatctgg tcccggacct tcggctcggg 180
cgaggccacg gccgacccag tcgcacacgt ggagctggag cgggggcagc cgttcggcct 240
ggccgcgcaa cgggatgagt acgtgaagtg ccaggtgatc ccgcaggccg gtgagccgcg 300
tgagttcatt cccgaccgcc cggccaacag cctgcgcagc tcgcggctgc ccgggccgga 360
acccgcgtgg ttctccggcc cggccgaggc ccgctgcacc gggcccacca cggtgctcct 420
gccggagcgc tacgacacga ccgggctgaa cgtcctgctg gggctgattg ctgcggccag 480
cctcatgatc gtggtcgttt ttgtacaatt cgcgcgtcgt gcccgtttca acaataggtt 540
gtacgcccgc gactgatttg ctcctgggaa cggatctccg aacggcatga ggcgccgtgg 600
gcggtcagat agtgtcgatc caacggttcg gaagctaagg ggacagacgg tgagcagcgc 660
gtctgagcgc atccaggaga tgatgcgcaa gttcgaggag caggcgcaga aggcgtcgga 720
actgcagtcc gcgatgcagg gcatgcaggc cacggcgagc agcccggacc ggtcggtgac 780
ggtgacggtc gcgccgtccg gggcggtgct ggacctgcgt ctggcgccga acgcggtgcg 840
ccagtcggcc aacgacctgc agcagcagat catggcgacc atccgccaag caacggcaag 900
cgcggcggag cagctcaaca acacggtggc gccgatcctg ggcgaccagt tcgacaagtt 960
ccaggaagcg ttcaacgtcg aaggcatggc gatcaagccg accgccggag acgaaacgcc 1020
cgcaccggag agccccgcac cgccgcccgc cgcgcaccgc gcgccgccgg agagtccggc 1080
aacgccgcgg cggccgcagc aggcgagcgc cgattacgac ggtgacgact tctcttcggg 1140
ctcgttcctg cgctgaggga agttgtcttg ac 1172

Claims (7)

1. a kind of carry out the method that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination, it is characterised in that: including Following steps:
(1) ura4 must found as negative selection marker gene in saccharopolyspora strain genome；
(2) sequence that design separately designs that one section of size is 1 kb at the both ends of ura4 gene is used to carry out ura4 gene replacement Homology arm；
(3) ura4 gene upstream and downstream homology arm is merged with A Bola resistant gene using fusion DNA vaccine, and and plasmid POJ260 is attached, and obtains recombinant plasmid pOJ260-ura4；
(4) protoplast containing recombinant plasmid pOJ260-ura4 is transferred in palpus saccharopolyspora strain, carries out striking for ura4 gene It removes, ura4 deletion mycopremna S.pogona- Δ ura4 is obtained, by linear fragment homologous recombination technique to the ura4 deletion mycopremna S.pogona- Δ ura4 carries out the blocking of palpus saccharopolyspora strain gene cluster.
2. according to claim 1 carry out the method that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination, It is characterized by: ura4 gene order described in step (1) is as shown in sequence table SEQ ID № 1.
3. according to claim 1 or claim 2 carry out the method that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination, It is characterized in that, for replacing upstream homology arm sequence such as 2 institute of sequence table SEQ ID № of ura4 gene described in step (2) Show；The downstream homology arm sequence for replacing ura4 gene is as shown in sequence table SEQ ID № 3.
4. being carried out described according to claim 1~one of 3 based on linear fragment homologous recombination must the blocking of saccharopolyspora strain gene cluster Method, which is characterized in that replace ura4 gene using apramycin resistance gene described in step (3), making must the more spore of sugar Bacterium has apramycin resistance, in the subsequent saccharopolyspora strain progress transformant screening to palpus, adds apramycin.
5. carrying out the side that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination described in one of Claims 1 to 4 Method, which is characterized in that step (4) obtains ura4 deletion mycopremna S.pogona- Δ ura4 and carries out as palpus saccharopolyspora strain gene cluster The type strain that large fragment blocks.
6. according to claim 5 carry out the method that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination, It is characterized in that, step (4) the linear fragment homologous recombination technique that passes through is to the ura4 deletion mycopremna S.pogona- Δ Ura4 carries out the blocking of palpus No. 14 gene clusters of saccharopolyspora strain, comprising the following steps:
(a) blacked-out areas downstream is waited in No. 14 gene clusters, genome is upper to be set to 2,627,531~2,631,482, designs two pairs Primers F c14up/Rc14up and Fc14down/Rc14down carry out the amplification of upstream and downstream homology arm, for linear fragment to be inserted into In genome；
(b) in No. 14 gene cluster region upstreams to be knocked out, genome is upper to be set to 2,609,020~2,609,519, and design is a pair of Primers F ds/Rds carries out direct sequence amplification, knocks out outside the external source ura4 gene ring for that will introduce；
(c) pair of primers Fura4/Rura4 progress ura4 gene magnification must be being designed in saccharopolyspora strain genome, as negative selection Marker gene carries out transformant screening；
(d) using plasmid pOJ260-cm-Perm* as template, design pair of primers Fperm/Rperm is used for erythromycin strong promoter (Perm*) it expands, for starting ura4 gene expression；
(e) genetic fragment that step (a) to step (d) obtains is merged using fusion DNA vaccine, acquisition can be used for No. 14 genes The linear recombinant fragment UAc14-Perm*-ura4-DS-DAc14 that cluster blocks；
(f) the linear recombinant fragment of protoplast transformation, which enters in S.pogona- Δ ura4, is recombinated, is screened, and No. 14 bases are obtained The engineered strain S. pogona- Δ ura4- Δ c14 blocked by cluster.
7. according to claim 6 carry out the method that palpus saccharopolyspora strain gene cluster blocks based on linear fragment homologous recombination, It is characterized in that, the engineered strain S. pogona- Δ ura4- Δ that resulting palpus No. 14 gene clusters of saccharopolyspora strain of step (f) block The blacked-out areas of c14 is 20 kb sequences.
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