CN107760642A - A kind of energy efficient-decomposition formamide and the recombination bacillus coli and its construction method of phosphite oxide and application - Google Patents

A kind of energy efficient-decomposition formamide and the recombination bacillus coli and its construction method of phosphite oxide and application Download PDF

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CN107760642A
CN107760642A CN201710904085.XA CN201710904085A CN107760642A CN 107760642 A CN107760642 A CN 107760642A CN 201710904085 A CN201710904085 A CN 201710904085A CN 107760642 A CN107760642 A CN 107760642A
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formamide
bacillus coli
petduet
ptx
phosphite
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娄文勇
区晓阳
李慧娴
宗敏华
徐培
魏萍
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South China University of Technology SCUT
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Abstract

The invention discloses a kind of energy efficient-decomposition formamide and the recombination bacillus coli and its construction method of phosphite oxide and application.The present invention is using bacillus coli DH 5 alpha engineering bacteria as host, obtained formamidase gene will be cloned and phosphorous dehydrogenase gene is connected in turn on carrier and obtains recombinant plasmid, and be transformed into E. coli expression strains BL21 (DE3), Fiber differentiation obtains recombination bacillus coli.The recombination bacillus coli of the present invention contains formamidase gene and phosphorous dehydrogenase gene simultaneously, can express formamidase simultaneously and decompose formamide generation NH4 +With expression phosphorous acidohydrogenase oxidation of phosphite phosphate, nitrogen source and phosphorus source required for growth and breeding is provided for restructuring colibacillus engineering, and other microorganisms do not possess this two acquisition route of nutrition, recombination bacillus coli is set to turn into dominant bacteria, solve the problems, such as Escherichia coli microbiological contamination in industrial fermentation, while reduce Zymolysis Equipment demand and reduce the addition of antibiotic.

Description

It is a kind of can efficient-decomposition formamide and phosphite oxide recombination bacillus coli and its Construction method and application
Technical field
The present invention relates to genetic engineering field, and in particular to a kind of weight of energy efficient-decomposition formamide and phosphite oxide Group Escherichia coli and its construction method and application.
Background technology
In recent years, developing rapidly with fermentation industry, fermentation technique, Zymolysis Equipment continue to develop renewal, in fermentation tube Reason experience is increasingly abundanter, and in the case of technological means maturation all the more, the fermentation contamination rate of each enterprise also has decline.But industry hair Microbiological contamination phenomenon still happens occasionally during ferment, once and microbiological contamination, waste of raw materials is not only caused, and production effect can be influenceed Rate, it is bothersome laborious, destroy the production schedule, upset production order etc..So the microbiological contamination that solve in fermentation remains fermentation enterprise One of the problem of most deeply concerned.Fermentation process is typically the purebred incubation of microorganism, only allows specific micro- life in incubation Thing grows, if also having other microorganisms to exist in addition to purpose bacterium in fermentation process, is treated as microbiological contamination.Microbiological contamination has a strong impact on mesh Bacterium growth, cause fermentation fail.Cause to ferment microbiological contamination the reason for it is a lot, the reason for except in manual operation, equipment causes Fermentation microbiological contamination account for significant portion.
Research on microbiological contamination of fermenting at present focuses mostly in the bacterium shape of the source (approach) of fermentation microbiological contamination and correlation, different hairs Influence and prophylactico-therapeutic measures of the microbiological contamination of ferment stage to fermentation, the analysis of slave unit angle and preventing and treating etc..Enterprise ferment to drop Low contamination rate, mainly start with from the maintenance and processing of Zymolysis Equipment, such as control fermentation tank air inlet system and water circulation System aseptic filtration is handled, it is ensured that steam sterilization system sterilizing is thorough, yeasting health and material processing, the purity of strain Deng.But microorganism is ubiquitous, being particularly some has microorganisms of spore structure, and moise-heat sterilization is possible can not be them " kill ".So have the pollution of miscellaneous bacteria unavoidably in Fermentation Engineering.
For microbiological contamination phenomenon, researcher is seldom studied from the intake of nutriment substrate spectrum, and this research is with large intestine bar Bacterium host cell is as research object, because Escherichia coli are to use most extensive, most successful expression system.To Escherichia coli Nutrition and Metabolism approach is transformed, and improved colibacillus engineering is decomposed formamide and is produced ammonia as nitrogen source and decomposition Phosphite oxide turns into phosphate as phosphorus source, changes the available sources of its nitrogen source and phosphorus source, it is trained in specific MOPS Energy normal growth in base is supported, without having the miscellaneous bacteria microorganism of this two metabolic pathways will be due to lacking nitrogen source or phosphorus source simultaneously The source of nutriment and by " hungry to death " so that the colibacillus engineering transformed turn into defined medium dominant bacteria, Miscellaneous bacteria cannot growth and breeding.The application of engineered Escherichia coli advantage engineering bacteria, on the one hand ensure engineering bacteria normal growth The target gene simultaneously on expression plasmid is bred, fermentation life on the other hand can also be carried out in unpasteurized, open fermentation tank Production, and miscellaneous bacteria is unable to growth and breeding, and reach the purpose of pure culture.
In molecular biology, the recombination and expression techniques of gene are quite ripe, can utilize the means of genetic engineering The gene of separate sources is realized and co-expressed in same Escherichia coli, possesses other bacterial strains so as to assign Escherichia coli Some special metabolic functions, can be more widely used in fermentation industry, reduce production cost and equipment requirement.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of energy efficient-decomposition formamide and oxidation Asia Phosphatic recombination bacillus coli.The recombination bacillus coli contains formamidase (FOR) gene and phosphorous acidohydrogenase simultaneously (PTDH) gene, formamidase can be expressed simultaneously and decomposes formamide generation NH4 +With expression phosphorous acidohydrogenase phosphite oxygen Change phosphate, the nitrogen source and phosphorus source required for growth and breeding are provided for restructuring colibacillus engineering, and have when other are different The microorganism of this two metabolic pathways then can be due to lacking the metabolic pathway of nitrogen source and phosphorus source and by " hungry to death ".
The present invention also aims to provide the restructuring of described a kind of energy efficient-decomposition formamide and phosphite oxide The construction method of Escherichia coli.The construction method will be cloned from series bacillus using bacillus coli DH 5 alpha engineering bacteria as host Paenibacillus pasadenensis.CS0611 formamidase (FOR) gene and Klebsiella Pneumoniae Klebsiella Pneumonia.OU7 phosphorous acidohydrogenase (PTDH) gene, it is connected to carrier pETDuet-1 two multiple cloning sites On, and obtained recombinant plasmid pETDuet-for-ptx is transformed into DH5 α, then the recombination bacillus coli DH5 α that will be obtained (pETDuet-for-ptx) it is transformed into E. coli expression strains E.coliBL21 (DE3), obtains recombinant strains BL21 (DE3) (pETDuet-for-ptx), and continue the Fiber differentiation in the MOPS culture mediums containing formamide and phosphite and recombinate Escherichia coli.
Series bacillus Paenibacillus pasadenensis.CS0611 containing formamidase gene and contain Asia The Klebsiella Pneumoniae Klebsiella pneumonia.OU7 of phosphate dehydrogenase gene provide for structure recombination bacillus coli Corresponding enzyme gene, the enzyme gene from different strains is amplified then two enzyme genes structure existed by PCR means In same colibacillus engineering, two enzyme genes are expressed, play its enzymatic function, realize that recombination bacillus coli exists Normal growth is bred and is expressed its function in MOPS culture mediums containing formamide and phosphite.
The present invention also aims to provide the application of described a kind of energy efficient-decomposition formamide and phosphite oxide.
The purpose of the present invention is achieved through the following technical solutions.
A kind of construction method of the recombination bacillus coli of energy efficient-decomposition formamide and phosphite oxide, including following step Suddenly：
(1) primer, PCR amplification formamidase genes are designed
Sense primer A1 (5'-CGCGATGAACGGACTGGGCGGCTTGAAC-3'), underscore italic is BamH I digestion points；Anti-sense primer A2 (5'-CCGCGTCGCGCCGCGCCTCCCTTCGCTC-3'), underscore is oblique Body is EcoR I digestion points；
Using series bacillus Paenibacillus pasadenensis.CS0611 genome as template, clone 1011bp formamidase gene order, is named as；The gene order for that clone obtains is connected to carrier pETDuet-1 On first multiple cloning sites, then obtained recombinant vector pETDuet-for is transformed into bacillus coli DH 5 alpha, contained Recombinant vector pETDuet-for recombination bacillus coli DH5 α (pETDuet-for)；
(2) primer, PCR amplification phosphorous dehydrogenase genes are designed
Sense primer B1 (5'-GAGATGCTGCCGAAACTCGTTATAACTC-3'), underscore italic is The digestion points of Bgl II；Anti-sense primer B2 (5'-GGACATGCGGCAGGCTCGGCCTTGGGC-3'), underscore is oblique Body is the digestion points of Kpn I；
Using Klebsiella Pneumoniae Klebsiella pneumonia.OU7 genomes as template, 1008bp phosphorous is cloned Dehydrogenase gene sequence, is named as ptx；The gene order ptx that clone obtains is connected to more than second of pETDuet-for On cloning site, formamidase gene for and phosphorous dehydrogenase gene ptx while the weight for being connected to same expression plasmid are obtained Group plasmid, is named as pETDuet-for-ptx；Recombinant plasmid pETDuet-for-ptx is transformed into DH5 α, obtains recombinating large intestine Bacillus DH5 α (pETDuet-for-ptx)；
(3) translation table reaches recombinant bacterial strain
The extraction recombinant plasmid pETDuet-for-ptx from recombination bacillus coli DH5 α (pETDuet-for-ptx), and will Recombinant plasmid pETDuet-for-ptx is transformed into E. coli expression strains BL21 (DE3), obtains recombinant strains BL21 (DE3)(pETDuet-for-ptx)；
(4) Fiber differentiation recombination bacillus coli
Obtained recombinant strains BL21 (DE3) (pETDuet-for-ptx) is inoculated into LB culture mediums, lured After leading culture, collect thalline and be added in the MOPS culture mediums containing formamide and phosphite, continue Fiber differentiation restructuring Escherichia coli.
Further, in step (1), the series bacillus Paenibacillus pasadenensis.CS0611's Formamidase gene order is as shown in sequence table SEQ 1, fragment length 1011bp, encodes 337 amino acid.
Further, in step (1), the series bacillus Paenibacillus pasadenensis.CS0611 in It is preserved in China typical culture collection center on October 8th, 2014, preserving number is CCTCC NO:M2014458.
Further, in step (1), the condition of the PCR amplifications is：94 DEG C of reaction 5min；10s, 55 are reacted in 98 DEG C DEG C reaction 5s, 72 DEG C reaction 70s, and circulate 30 times；Then at 72 DEG C of reaction 7min；Finally it is cooled to 16 DEG C.
Further, in step (2), the phosphorous acid of the Klebsiella Pneumoniae Klebsiella pneumonia.OU7 takes off Hydrogenase gene sequence is as shown in sequence table SEQ 2, fragment length 1008bp, encodes 336 amino acid.
Further, in step (2), the Klebsiella Pneumoniae Klebsiella pneumonia.OU7 were in 2017 8 The moon is preserved in China typical culture collection center on the 24th, and preserving number is CCTCC NO.M2017449.
Further, in step (2), the condition of the PCR amplifications is：94 DEG C of reaction 5min；10s, 55 are reacted in 98 DEG C DEG C reaction 5s, 72 DEG C reaction 70s, and circulate 30 times；Then at 72 DEG C of reaction 7min；Finally it is cooled to 16 DEG C.
Further, in step (4), the Fiber differentiation be in LB culture mediums, 37 DEG C, 180rpm cultivate 12 hours Afterwards, it is inoculated in fresh LB culture mediums, 37 DEG C, 180rpm Fiber differentiations to restructuring bacteria concentration to OD600=0.6, it is cooled to After 20 DEG C, the isopropyl-β-D-thiogalactoside (IPTG) for adding final concentration of 0.2mM carries out induction 16h.
Further, in step (4), it is described collect thalline be bacterium solution after Fiber differentiation is terminated 4 DEG C, 8000rpm, Centrifuge 5min after, then be cooled in advance 4 DEG C physiological saline suspend, 4 DEG C again, 8000rpm, centrifugation 5min, repeatedly hanged with salt solution The step of floating and centrifuging twice, to remove the LB culture mediums of residual, collects thalline.
Further, in step (4), in the MOPS culture mediums containing formamide and phosphite, the end of formamide Concentration is 200mM, the final concentration of 1.32mM of phosphite.
Further, in step (4), the thalline of collection is added in the MOPS culture mediums containing formamide and phosphite Afterwards, cell concentration OD600=0.1.
Further, in step (4), the Fiber differentiation that continues is in the MOPS cultures containing formamide and phosphite Final concentration of 0.2mM IPTG is added in base, and continues culture 4 days in 30 DEG C, 180rpm.
Lack basal nutrient composition NH in MOPS culture mediums4 +And HPO4 2-And formamide and phosphite are added with, at the same it is miscellaneous Bacteria microorganism does not possess the function of decomposing formamide and phosphite oxide acquisition nutritional ingredient, miscellaneous bacteria microorganism is lacked nitrogen source And phosphorus source, make recombination bacillus coli due to obtaining the battalion of abundance with the efficient function of decomposing formamide and aoxidize phosphorous acid Form point and turn into dominant bacteria.
The restructuring of the energy efficient-decomposition formamide and phosphite oxide that build to obtain by any of the above-described methods described is big Enterobacteria, wherein, formamidase (FOR) gene and phosphorous acidohydrogenase (PTDH) gene are single carrier in Escherichia coli, The coexpression realized using double-promoter, coexpression vector pETDuet-for-ptx, and formamidase (FOR) gene and Asia Phosphate dehydrogenase (PTDH) gene all realizes solubility expression in the plasmid pGEX-2T containing GST labels respectively.
The recombination bacillus coli of obtained energy efficient-decomposition formamide and phosphite oxide is built, formyl can be expressed simultaneously Amine enzyme decomposes formamide generation NH4 +It is recombination bacillus coli with expression phosphorous acidohydrogenase oxidation of phosphite phosphate Normal growth breeding provides nitrogen source and phosphorus source, and other microorganisms possess this two approach due to lacking, thus can not containing Growth and breeding in the MOPS culture mediums of formamide and phosphite, so that recombination bacillus coli turns into dominant bacteria, prevents from recombinating Escherichia coli are in fermentation process is produced by living contaminants.
The recombination bacillus coli of described a kind of energy efficient-decomposition formamide and phosphite oxide is applied to Escherichia coli Industrial fermentation enzyme' s catalysis includes the valuable material of antibody or pharmaceutical intermediate.
Compared with prior art, the invention has the advantages that and beneficial effect：
(1) recombination bacillus coli of the invention can express formamidase simultaneously and decompose formamide generation NH4 +With expression phosphorous Acidohydrogenase provides nitrogen source and phosphorus source oxidation of phosphite phosphate, for the normal growth breeding of recombination bacillus coli, and its His microorganism possesses this two approach due to lacking, and can not be grown in the MOPS culture mediums containing formamide and phosphite numerous Grow, solve the problems, such as Escherichia coli microbiological contamination in industrial fermentation, while reduce Zymolysis Equipment demand and reduce antibiotic Addition；
(2) present invention solves the problems, such as the microbiological contamination during Escherichia coli fermentation, mesh by the biotechnology of genetic engineering Property it is strong, there is thorough validity；
(3) recombination bacillus coli of the invention, which is applied to Escherichia coli industrial fermentation enzyme' s catalysis, is included in antibody or medicine The valuable material of mesosome, enzyme' s catalysis is included the processing efficient of valuable material of antibody or pharmaceutical intermediate, close Into material purity it is high, no miscellaneous bacteria product pollution.
Brief description of the drawings
Fig. 1 is restructuring co-expression plasmid pETDuet-for-ptx building process figure；
Fig. 2 is growth curve of the restructuring e. coli bl21 (DE3) (pETDuet-for-ptx) in MOPS culture mediums Figure；
Fig. 3 utilizes formamide and phosphorous acid for restructuring e. coli bl21 (DE3) (pETDuet-for-ptx) assimilation Salt and as the advantage engineering bacteria procedure chart in culture medium.
Embodiment
To more fully understand the present invention, with reference to embodiments and accompanying drawing is further elaborated to technical solution of the present invention, But described content is merely to illustrate the present invention, without should not also limit the present invention.
The series bacillus Paenibacillus pasadenensis.CS0611 used in the specific embodiment of the invention in It is preserved in China typical culture collection center on October 8th, 2014, preserving number is CCTCC NO:M2014458, and completed Full-length genome is tested, and the fragment of formamidase gene therein is as shown in sequence table SEQ 1, length 1011bp, encodes 337 Amino acid.
Klebsiella Pneumoniae Klebsiella pneumonia.OU7 in the specific embodiment of the invention were in August 24 in 2017 Day is preserved in China typical culture collection center (Wuhan City, Hubei Province Wuchang road Luo Jia Shan Wuhan University, postcode 430072), Preserving number is CCTCC NO.M 2017449, and Klebsiella Pneumoniae Klebsiella pneumonia.OU7 are to pass through such as lower section Independently screening obtains for method culture, specifically comprises the following steps：
The culture medium used is Meng Jinna solid mediums (1L)：Glucose 10g, (NH4)2SO40.5g, MgSO4·H2O 0.3g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.0036g, MnSO4·H2O0.03g, CaSO4·2H2O 1g, KH2PO30.6g and agar 20g, ultra-pure water are settled to 1L.
(1) enrichment culture：Sampled in kitchen sewer water ditch, take 1mL samples, be added in 100mL sterilized water, fully shake It is even, bacteria suspension is made；1mL bacteria suspensions are taken to be linked into the 5mL enriched mediums for bacterium of having gone out in advance respectively, 37 DEG C of enrichment cultures 48h；
(2) flat board primary dcreening operation：Bacterium solution 10 after enrichment culture is diluted step by step again, it is 10 to take extension rate respectively-3、10-5、 10-7Bacteria suspension 0.5mL be coated on the Selective agar medium flat board containing phosphite, 37 DEG C of culture 48h will be longer larger Single bacterium colony choose into inclined-plane Meng Jinna solid mediums, it is so repeated multiple times, until being defined as pure bacterium, protected using slant medium Hide original strain.
The Klebsiella Pneumoniae Klebsiella pneumonia.OU7 independently screened 16S rDNA and NCBI has been announced Bacterial strain Klebsiella sp.F5-1 and Klebsiella sp.CCFM8375 compare bacterium be 99%.
The Klebsiella Pneumoniae Klebsiella pneumonia.OU7 independently screened are tested by full-length genome, therein The fragment of phosphorous dehydrogenase gene is as shown in sequence table SEQ 2, length 1008bp, encodes 336 amino acid.
The Klebsiella Pneumoniae Klebsiella pneumonia.OU7 independently screened phosphorous acid dehydrogenase gene sequence With NCBI websites disclosed in Klebsiella pneumonia strain AR, Klebsiella pneumonia strain MNCRE53 phosphorous dehydrogenase gene homology is 100%, the Asia with Pseudomonas stutzeri strain 273 The homology of phosphate dehydrogenase gene is 99%.
Embodiment 1
Formamidase gene for acquisition
By series bacillus Paenibacillus pasadenensis.CS0611 using LB culture mediums 37 DEG C, 180rpm is cultivated one day；Cultured thalline is collected into thalline in 4 DEG C, 8000rpm, centrifugation 5min, with brine 2 It is secondary, to remove the culture medium of residual, then according to the specific method of OMEGA bacterial genomes DNA extraction kits to class gemma Bacillus Paenibacillus pasadenensis.CS0611 genomes are extracted；
Using the Paenibacillus pasadenensis.CS0611 genomes of extraction as template, with A1 (5'-CGC) and A2 (5'-CCG GATGAACGGACTGGGCGGCTTGAAC-3' CGTCGCGCCGCGCCTCCCTTCGCTC-3' it is respectively) upstream and downstream primer, formamidase gene is expanded using PCR；
Enzymatic reagent used in PCR is TaKaRa companiesHS DNA Polymerase with GC buffer；PCR reaction systems and condition are as follows：
PCR reaction conditions：94 DEG C of reaction 5min；React 10s, 55 DEG C of reaction 5s, 72 DEG C of reaction 70s in 98 DEG C successively again, And circulate 30 times；Most 7min is reacted after 72 DEG C.
The PCR DNA products for expanding to obtain are utilized into 1wt% agarose gel electrophoresis, and use OMEGA companies glue reclaim reagent Box, to specifications step progress gel extraction, then censorship are sequenced, as a result show and obtained the first that a segment length is 1011bp Amidase gene sequence, is named as.
Embodiment 2
Phosphorous dehydrogenase gene ptx acquisition
Phosphorous dehydrogenase gene derives from the Klebsiella Pneumoniae Klebsiella pneumonia.OU7 independently screened, By independently screening to obtain, screening and culturing medium uses Meng Jinna solid mediums (g)：Glucose 10, (NH4)2SO40.5, MgSO4·7H2O 0.3, NaCl 0.3, KCl 0.3, FeSO4·7H2O 0.0036, MnSO4·H2O 0.03, KH2PO30.3, Agar 20,1L is settled to ultra-pure water；
The single bacterium colony grown in screening flat board is dipped with transfer needle and, then proceedes to enter in new screening flat board Row classification line, until there is single bacterium colony, reaches purifying, is finally recovered and is purified into one plant growing way is very on this screening and culturing medium One plant of good bacterial strain, is sequenced by 16S rDNA, is as a result shown as Klebsiella Pneumoniae Klebsiella pneumonia.OU7.
According to the specific method of OMEGA bacterial genomes DNA extraction kits to the Klebsiella Pneumoniae that independently screens Klebsiella pneumonia.OU7 genomes are extracted, with the Klebsiella Pneumoniae Klebsiella independently screened Pneumonia.OU7 genome is template, with B1 (5'-GAGATGCTGCCGAAACTCGTTATAACTC-3') With B2 (5'-GGACATGCGGCAGGCTCGGCCTTGGGC-3' it is respectively) upstream and downstream primer, is expanded using PCR Phosphorous dehydrogenase gene；
Enzymatic reagent used in PCR is TaKaRa companiesHS DNA Polymerase with GC buffer；PCR reaction systems and condition are as follows：
PCR reaction conditions：94 DEG C of reaction 5min；React 10s, 55 DEG C of reaction 5s, 72 DEG C of reaction 70s in 98 DEG C successively again, And circulate 30 times；Most 7min is reacted after 72 DEG C.
The PCR DNA products for expanding to obtain are utilized into 1wt% agarose gel electrophoresis, and use OMEGA companies glue reclaim reagent Box, to specifications step progress gel extraction, then censorship are sequenced, as a result show and obtained the Asia that a segment length is 1008bp Phosphate dehydrogenase gene sequence, is named as ptx.
Embodiment 3
Recombinant bacterium BL21 (DE3) (pETDuet-for) structure
The genetic fragment for obtained in embodiment 1 and plasmid pETDuet-1 are utilized respectively into BamH I and EcoR I to carry out Double digestion, sense primer underscore italic are BamH I digestion points, and anti-sense primer underscore italic is EcoR I digestion points, digestion Condition is：37 DEG C, digestion 60min；
Product after digestion is subjected to the glue reclaim examination of gel extraction, recovery method and step with reference to OMEGA companies respectively Agent box；1wt% agarose gel electrophoresis are utilized after recovery, detect the rate of recovery；Then the purpose fragment of recovery and plasmid are connected Connect, connect the T4DNA Ligase that kit uses Thermo Fisher SCIENTIFIC companies, linked system is as follows：
Purpose fragment is 3 with plasmid mol ratio：1.
Connection product is transformed into bacillus coli DH 5 alpha, step of converting is as follows：By 10 μ L connection products and 100 μ L large intestine bars Bacterium DH5 α competent cells mix, ice bath 30min, 42 DEG C of heat shock 90s, then ice bath 2min, 890 μ L LB culture mediums of addition, and 37 DEG C, after 180rpm shaking table cultures 1h, 4000rpm is centrifuged 5 minutes and is collected thalline, the μ L of supernatant culture medium 890 is taken, by the thalline of ttom of pipe It is resuspended, is uniformly coated to the resistance element LB of benzyl containing ammonia solid plates (containing 100 μ g/mL ampicillin sodiums), 37 DEG C of cultures 16 is small When, after flat board grows transformant, picking transformant enters performing PCR checking and censorship sequencing, and using DNAssist softwares to surveying Sequence result carries out ORF lookups.
As a result show, obtain first polyclonal position that formamidase gene order for has been correctly inserted into pETDuet-1 On point, pETDuet-for plasmids are have successfully been obtained, and are transformed into BL21 (DE3), obtain recombinant bacterium BL21 (DE3) (pETDuet- for)。
Embodiment 4
Recombinant bacterium BL21 (DE3) (pETDuet-for-ptx) structure
The phosphorous dehydrogenase gene ptx that embodiment 2 obtains and the pETDuet-for that embodiment 3 obtains are utilized respectively Bgl II and Kpn I carries out double digestion, and sense primer underscore italic is the digestion points of Bgl II, and anti-sense primer underscore italic is Kpn I digestion points, digestion condition are：37 DEG C, digestion 60min；
Digestion system, recovery method and connection method for transformation are same as Example 3, specific recombinant plasmid pETDuet-for- Ptx building process figure as shown in figure 1, formamidase gene is first subcloned into first cloning site of plasmid pETDuet-1, Obtain recombinant plasmid pETDuet-for.Second segment phosphorous dehydrogenase gene is cloned into pETDuet-for more than second again Cloning site, obtain recombinant plasmid pETDuet-for-ptx.
Picked clones enters performing PCR checking, and censorship is sequenced；As a result show, phosphorous dehydrogenase gene ptx has succeeded Ground connects second multiple cloning sites on pETDuet-for, has obtained recombinant plasmid pETDuet-for-ptx, and weight Group plasmid is transformed into BL21 (DE3), and obtained bacterial strain is recombination bacillus coli BL21 (the DE3) (pETDuet- to be built for-ptx)。
Embodiment 5
Growths of the recombination bacillus coli BL21 (DE3) (pETDuet-for-ptx) in specific MOPS culture mediums
The recombinant strains BL21 (DE3) (pETDuet-for-ptx) of acquisition is subjected to Fiber differentiation, detailed process is such as Under：
By BL21 (DE3) (pETDuet-for-ptx) according to volume ratio 1：100 are inoculated into the LB culture mediums containing 50mL, 37 DEG C, 180rpm is incubated overnight 12 hours；Fiber differentiation is carried out, the recombination bacillus coli for taking 1mL to be incubated overnight, which is inoculated into, to be equipped with LB culture mediums fresh 100mL, in 37 DEG C, 180rpm cultures restructuring bacteria concentration to OD600=0.6, after being cooled to 20 DEG C, add After final concentration of 0.2mM IPTG carries out induction 16h, 4 DEG C, 8000rpm, centrifugation 5min collect thalline；
Suspended the thalline collected with physiological saline (4 DEG C of precoolings), 4 DEG C, 8000rpm, centrifugation 5min collection thalline, and repetition This step 2 time, remove the LB culture mediums of residual；Thalline is added to containing formamide (200mM) and phosphite again The basic MOPS culture mediums of (1.32mM), make thalline OD600For 0.1, final concentration of 0.2mM IPTG, 30 DEG C, 180rpm are added Cultivate recombination bacillus coli.
Investigate growing state of the recombinant bacterium in the MOPS culture mediums containing formamide and phosphite, recombinant bacterium BL21 (DE3) life of (pETDuet-for-ptx) in the MOPS culture mediums containing formamide (200mM) and phosphite (1.32mM) Long curve map is as shown in Fig. 2 recombinant bacterium BL21 (DE3) (pETDuet-for-ptx) can give birth in the culture medium as shown in Figure 2 Long, to after the 3rd day, bacteria concentration reaches maximum, A600For 1.321；And control strain BL21 (DE3) is in the MOPS culture medium bases This does not grow.
Recombination bacillus coli BL21 (DE3) (pETDuet-for-ptx) assimilation using formamide and phosphite and As the advantage engineering bacteria procedure chart in culture medium as shown in figure 3, e. coli bl21 (DE3) (pETDuet-for-ptx) by In inserting formamidase gene and phosphorous dehydrogenase gene, so improved e. coli bl21 (DE3) (pETDuet- For-ptx formamide can) be decomposed into NH4 +, and can phosphite oxide is into phosphate, so as to be e. coli bl21 (DE3) (pETDuet-for-ptx) provides the nitrogen source and phosphorus source that growth utilizes, and other miscellaneous bacteria microorganisms gather around simultaneously due to lacking There are this two approach, so can not grow.
MOPS culture mediums lack NH4 +And HPO4 2-, but with the addition of formamide and phosphite and come as nitrogen source and phosphorus source Source, recombination bacillus coli BL21 (DE3) (pETDuet-for-ptx) can express formamidase and phosphorous acidohydrogenase, formamide Enzyme, which can decompose formamide, turns into NH4 +, while phosphorous acid dehydrogenase oxidoreductase phosphite turns into phosphate, is recombinant bacterium BL21 (DE3) nutriment (nitrogen source and phosphorus source) necessary to the breeding of (pETDuet-for-ptx) own growth provides, and other microorganisms Due to it is different when there are this two metabolic pathways, it is therein a kind of or same that two kinds of nutriments (nitrogen source and phosphorus source) will be lacked When lack two kinds of nutriments and by " hungry to death ".
Sequence table
<110>South China Science ＆ Engineering University
<120>A kind of energy efficient-decomposition formamide and the recombination bacillus coli and its construction method of phosphite oxide and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1014
<212> DNA
<213>Series bacillus (Paenibacillus pasadenensis. CS0611)
<400> 1
atgaacggac tgggcggctt gaacaaatcg ccggacggcg tcgtcatcgg cctcgcgcag 60
ctgcagctgc cggccgtcga gacgccggag cagctggcgg cgcaggcgag gcgcatcgcg 120
gagatgacgg ccaaggcgcg gaagggctcg cgctcgatgg atctgatcgt atttcccgaa 180
tattcgctgc atggcctatc gatgaatacc gatccggcgc tcatgtgccg ggtcgacggg 240
ccggaggtcg agctgtggcg ggaggcctgc cgcgagcatc gcatctgggg ctgcttcagc 300
atcatggagc tcaatccgga cggcaatccg tacaacacgg ggctcatcat cgacgacgaa 360
ggcggaatcc ggctgaagta ccgcaagctg catccgtggg tgccggtgga gccgtgggag 420
ccgggcgatc tcggcatccc gatgtgcgac gggccgaacg gcagccggtt ggcgctcgtc 480
atctgccatg acggcatgtt cccggaaatg gcgcgcgaat gcgcctatct cggtgcggac 540
atcatgctgc gcacggccgg ctatacggct ccgatccgcc atgcatggca ggtgacgaac 600
caggcgcacg ccttttgcaa cctgatgtac accgcctcgg tctgcctgag tggcagcgac 660
ggcgtcttcg actcgatggg cgaggcgatg atcgtcggct tcgacggcat gacgctcgtc 720
cacggcggcg gacggcccga cgagatcgtg gccggcgagg tgcggccgtc gctcgtccgc 780
gaggcgcgcc gcatctgggg cgtggagaac aacctgtacc agctcggcca tcgcggctac 840
gtcgcggtgc agggcggcgc aggcgactgt ccgtacacct acatgcatga tctcgccgcc 900
ggccgctacc ggctgccttg ggaggatgag gtgctcatca aggacggcac gagcgagggg 960
ttcccgccgc cggagcggcg atatggcgga gcgaagggag gcgcggcgcg atga 1014
<210> 2
<211> 1008
<212> DNA
<213>Klebsiella Pneumoniae (Klebsiella pneumonia. OU7)
<400> 2
atgctgccga aactcgttat aactcaccga gtacacgatg agatcctgca actgctggcg 60
ccacattgcg agctggtgac caaccagacc gacagcacgc tgacgcgcga ggaaattctg 120
cgccgctgtc gcgatgctca ggcgatgatg gcgttcatgc ccgatcgggt cgatgcagac 180
tttcttcaag cctgccctga gctgcgtgta gtcggctgcg cgctcaaggg cttcgacaat 240
ttcgatgtgg acgcctgtac tgcccgcggg gtctggctga ccttcgtgcc tgatctgttg 300
acggtcccga ctgccgagct ggcgatcgga ctggcggtgg ggctggggcg gcatctgcgg 360
gcagcagatg cgttcgtccg ctctggcgag ttccagggct ggcaaccaca gttctacggc 420
acggggctgg ataacgctac ggtcggcatc cttggcatgg gcgccatcgg actggccatg 480
gctgagcgct tgcagggatg gggcgcgacc ctgcagtacc acgaggcgaa ggctctggat 540
acacaaaccg agcaacggct cggcctgcgc caggtggcgt gcagcgaact cttcgccagc 600
tcggacttca tcctgctggc gcttcccttg aatgccgata cccagcatct ggtcaacgcc 660
gagctgcttg ccctcgtacg gccgggcgct ctgcttgtaa acccctgtcg tggttcggta 720
gtggatgaag ccgccgtgct cgcggcgctt gagcgaggcc agctcggcgg gtatgcggcg 780
gatgtattcg aaatggaaga ctgggctcgc gcggaccggc cgcggctgat cgatcctgcg 840
ctgctcgcgc atccgaatac gctgttcact ccgcacatag ggtcggcagt gcgcgcggtg 900
cgcctggaga ttgaacgttg tgcagcgcag aacatcatcc aggtattggc aggtgcgcgc 960
ccaatcaacg ctgcgaaccg tctgcccaag gccgagcctg ccgcatgt 1008

Claims (10)

  1. A kind of 1. construction method of the recombination bacillus coli of energy efficient-decomposition formamide and phosphite oxide, it is characterised in that Comprise the following steps：
    (1) primer, PCR amplification formamidase genes are designed
    Sense primer A1 (5'-CGCGGATCCGATGAACGGACTGGGCGGCTTGAAC-3'), underscore italic is BamH I enzymes Point of contact；Anti-sense primer A2 (5'-CCGGAATTCCGTCGCGCCGCGCCTCCCTTCGCTC-3'), underscore italic is EcoR I Digestion point；
    Using series bacillus Paenibacillus pasadenensis.CS0611 genome as template, 1011bp is cloned Formamidase gene order, be named as；To clone that obtained gene order for is connected on carrier pETDuet-1 the One multiple cloning sites, then recombinant vector is transformed into bacillus coli DH 5 alpha, obtain the weight containing recombinant vector pETDuet-for Group bacillus coli DH 5 alpha (pETDuet-for)；
    (2) primer, PCR amplification phosphorous dehydrogenase genes are designed
    Sense primer B1 (5'-GAAGATCTGATGCTGCCGAAACTCGTTATAACTC-3'), underscore italic is the enzymes of Bgl II Point of contact；Anti-sense primer B2 (5'-GGGGTACCACATGCGGCAGGCTCGGCCTTGGGC-3'), underscore italic is the enzymes of Kpn I Point of contact；
    Using Klebsiella Pneumoniae Klebsiella pneumonia.OU7 genome as template, 1008bp phosphorous acid is cloned Dehydrogenase gene sequence, it is named as ptx；The gene order ptx that clone obtains is connected to more than second grams of pETDuet-for On grand site, formamidase gene for and phosphorous dehydrogenase gene ptx while the restructuring for being connected to same expression plasmid are obtained Plasmid, it is named as pETDuet-for-ptx；Recombinant plasmid pETDuet-for-ptx is transformed into DH5 α, obtains recombinating large intestine bar Bacterium DH5 α (pETDuet-for-ptx)；
    (3) translation table reaches recombinant bacterial strain
    The extraction recombinant plasmid pETDuet-for-ptx from recombination bacillus coli DH5 α (pETDuet-for-ptx), and will restructuring Plasmid pETDuet-for-ptx is transformed into E. coli expression strains BL21 (DE3), obtains recombinant strains BL21 (DE3)(pETDuet-for-ptx)；
    (4) Fiber differentiation recombination bacillus coli
    Obtained recombinant strains BL21 (DE3) (pETDuet-for-ptx) is inoculated into LB culture mediums, carries out induction training After supporting, collect thalline and be added in the MOPS culture mediums containing formamide and phosphite, continue culture and obtain recombinating large intestine Bacillus.
  2. 2. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (1), the series bacillus Paenibacillus pasadenensis.CS0611's Formamidase gene order is as shown in sequence table SEQ 1, fragment length 1011bp, encodes 337 amino acid；The class gemma Bacillus Paenibacillus pasadenensis.CS0611 were preserved in China typical culture collection on October 8th, 2014 Center, preserving number are CCTCC NO:M2014458.
  3. 3. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (2), the phosphorous acid of the Klebsiella Pneumoniae Klebsiella pneumonia.OU7 Dehydrogenase gene sequence is as shown in sequence table SEQ 2, fragment length 1008bp, encodes 336 amino acid；The kerekou pneumonia Primary bacterium Klebsiella pneumonia.OU7 were preserved in China typical culture collection center, preservation on 24th in August in 2017 Number it is CCTCC NO.M 2017449.
  4. 4. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (1), (2), the condition of the PCR amplifications is：94 DEG C of reaction 5min；In 98 DEG C of reactions 10s, 55 DEG C of reaction 5s, 72 DEG C of reaction 70s, and circulate 30 times；Then at 72 DEG C of reaction 7min；Finally it is cooled to 16 DEG C.
  5. 5. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (4), the Fiber differentiation be in LB culture mediums, 37 DEG C, 180rpm cultivate 12 hours Afterwards, it is inoculated in fresh LB culture mediums, continues at 37 DEG C, 180rpm cultivated to restructuring bacteria concentration to OD600=0.6, cooling To after 20 DEG C, the isopropyl-β-D-thiogalactoside for adding final concentration of 0.2mM carries out induction 16h.
  6. 6. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (4), it is described collect thalline be bacterium solution after Fiber differentiation is terminated 4 DEG C, 8000rpm, centrifugation 5min after, then use the salt aqueous suspension for being cooled to 4 DEG C in advance, 4 DEG C again, 8000rpm, centrifuge 5min, repetition salt Aqueous suspension and the step of centrifuge twice, collect thalline.
  7. 7. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (4), in the MOPS culture mediums containing formamide and phosphite, the end of formamide Concentration is 200mM, the final concentration of 1.32mM of phosphite.
  8. 8. the structure of the recombination bacillus coli of a kind of energy efficient-decomposition formamide according to claim 1 and phosphite oxide Construction method, it is characterised in that in step (4), the thalline of collection is added to the MOPS culture mediums containing formamide and phosphite In after, cell concentration OD600=0.1；It is described that to continue culture be the isopropyl-β-D- that add final concentration of 0.2mM in the medium Thiogalactoside, and continue culture 4 days in 30 DEG C, 180rpm.
  9. 9. a kind of energy efficient-decomposition formamide and oxidation phosphorous acid that build to obtain by any one of claim 1~8 methods described The recombination bacillus coli of salt.
  10. 10. the recombination bacillus coli of a kind of the energy efficient-decomposition formamide and phosphite oxide described in claim 9 is applied to Escherichia coli industrial fermentation enzyme' s catalysis includes the valuable material of antibody or pharmaceutical intermediate.
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