CN105368838B - The product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate and its application - Google Patents
The product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate and its application Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/30—Wastewater or sewage treatment systems using renewable energies
- Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy
Abstract
The invention discloses the product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate, for product phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) JN459 M571, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 1st, 2015, deposit number is CGMCC NO.11770.The present invention is mutated the promoter of ppk2 genes using continuous error-prone PCR method in vitro, using JN459 to go out bacterium germination, strain storehouse containing ppk2 gene promoter mutant is established by gene recombination technology, screening obtains the ability decline for decomposing polyphosphate under anaerobic, and JN459 M571 bacterial strain of the ability without significant difference of polyphosphate is synthesized under aerobic condition.Bacterial strain of the present invention is 2.4 times higher than the Poly P contents of original strain JN459 in anaerobism operational process, can be applied to the EBPR Biological Phosphorus Removal Processes of sewage treatment plant.
Description
Technical field
The invention belongs to Microbial Breeding and biological technical field, and in particular to the product of one plant of efficient accumulation polyphosphate
Phosphorus solar month of 30 days bacterium engineering bacteria and its application.
Background technology
Current body eutrophication problem is quite serious, research confirm phosphorus be cause body eutrophication key factor it
One, therefore the phosphorus element content reduced in sewage disposal plant effluent is an important measures for controlling body eutrophication.At present
Sewage treatment plant generally using enhanced Biological Phosphorus Removal System (Enhance biological phosphorus removal,
EBPR) as preferred biological phosphate-eliminating technology.The realization of EBPR biological phosphorus removal functionals depends on poly- phosphorus microorganism in activated sludge
The effect of poly- phosphorus, poly- phosphorus microorganism is a kind of microorganism general name that can synthesize polyphosphate, is had in excessive consumption water body
Titanium pigment and the characteristics of be stored in thalline with polyphosphoric acids salt form.
Product phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) is a representative for poly- phosphorus microorganism, what it was accumulated
Polyphosphate (Poly-P) can reach more than the 10% of dry cell weight, have good phosphor-removing effect in EBPR systems.But
It is as other poly- phosphorus microorganisms, product phosphorus solar month of 30 days bacterium is there are a synthesis and the circulation of decomposition polyphosphate, i.e., aerobic
When synthesize polyphosphate, polyphosphate is decomposed when anaerobism to obtain energy, maintains thalline to GTP and ATP
Demand.This characteristic of product phosphorus solar month of 30 days bacterium have impact on its effect in EBPR, particularly when supplying oxygen not occur in EBPR systems
When sufficient water outlet total phosphorus content can be caused to reach legal discharge standard.Therefore, a kind of decomposition under anaerobic of exploitation is more
The product phosphorus solar month of 30 days bacterium that Quadrafos ability is greatly lowered has very important actual application value.
Polyphosphate kinase PPK2 is the key of reversible reaction between catalysis product phosphorus solar month of 30 days bacterium Poly-P and ATP/GTP
Enzyme.Cellular energy is sufficient under aerobic condition, and titanium pigment is catalyzed and synthesized Poly-P, anaerobism by PPK2 under the driving of ATP/GTP
Under the conditions of PPK2 catalysis Poly-P be decomposed to form ATP/GTP, maintain the nucleotide storehouse of cell to balance, meet the normal growth of cell
Demand.The special role of PPK2, which determines, to transform product phosphorus solar month of 30 days bacterium by the way of ppk2 genes are inactivated, at present
Also there has been no on obtaining the report that high-performance accumulates phosphorus solar month of 30 days bacterium by transforming ppk2 gene promoters.
The content of the invention
For the above-mentioned prior art, the object of the present invention is to provide the product phosphorus solar month of 30 days bacterium of one plant of efficient accumulation polyphosphate
Engineering bacteria and its application.By transforming the promoter of ppk2 genes, weaken activation of the hypoxia condition to ppk2 genes, reduce
The expression of ppk2, reduces decomposition of the PPK2 to Poly-P, the final performance for improving product phosphorus solar month of 30 days bacterium accumulation Poly-P.
To achieve the above object, the present invention adopts the following technical scheme that：
One aspect of the present invention is related to a kind of ppk2 gene promoters of oriented transformation, it has such as SEQ ID
Nucleotide sequence shown in NO.1, or through replacing, lacking or add, one or several bases are formed the sequence has equal work(
The nucleotide sequence of energy.
Another aspect of the present invention is related to the promoter reduces application in ppk2 expressions under low oxygen conditions.
The further aspect of the present invention is related to a kind of long-pending phosphorus solar month of 30 days bacterium, it contains the ppk2 gene promoters of above-mentioned oriented transformation
Son.
Another aspect of the invention is related to the product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate, bacterial strain tool
Body is product phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) JN459-M571.In on December 1st, 2015 is preserved in
(abbreviation CGMCC, address are BeiChen West Road, Chaoyang District, BeiJing City to state's Microbiological Culture Collection administration committee common micro-organisms center
No. 1 institute 3, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.11770.
Product phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) JN459-M571 of the present invention and wild type product phosphorus are small
Month bacterium JN459 is compared, and ppk2 gene promoters have the change of 3 bases, is -46, -76 and -79 bit bases respectively all by G
Become A.
It is a further object of the present invention to provide a kind of microbial inoculum, the active ingredient of the microbial inoculum is product phosphorus solar month of 30 days bacterium
(Microlunatus phosphovorus)JN459-M571。
Long-pending phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) JN459-M571 and/or the microbial inoculum are being made
Application in standby biological phosphate-eliminating agent falls within protection scope of the present invention.
The present invention a further object is that providing a pair is used for the easy of directional transformation (i.e. external mutation) ppk2 gene promoters
The primer of wrong PCR, its primer sequence is respectively as shown in SEQ ID NO.2 in sequence table and SEQ ID NO.3.
It is also another object of the present invention to provide product phosphorus solar month of 30 days bacterium (Microlunatus phosphovorus) JN459-
The preparation method of M571, is in vitro mutated the promoter of ppk2 genes using continuous error-prone PCR method, with existing long-pending phosphorus
Solar month of 30 days, bacterium JN459 was bacterium germination, establishes the strain storehouse containing ppk2 gene mutation promoters by homologous recombination technique, measures
The change of Poly-P contents under oxygen and anaerobism service condition, screening obtain high-performance product phosphorus solar month of 30 days bacterium JN459-M571.
Sense primer ppk2ProForward sequences：GTCGAAATGTCGGAAGCCGTGC(SEQ ID NO.2)；
Anti-sense primer ppk2ProReverse sequences：TAACCCATTGTTCGCCCGGCAG(SEQ ID NO.3).
The specific method that the continuous error-prone PCR method is mutated ppk2 gene promoters in vitro is：
(1) PCR reaction systems：1~3ul of template DNA 2ul, PCR buffer solution 5ul, Taq archaeal dna polymerase 1ul, dATP,
1~3ul of dTTP, 1~3ul of dGTP, 1~3ul of dCTP, sense primer 2ul, anti-sense primer 2ul, 25mM MgCl21~8ul,
5mM MnCl20~1ul, adds ultra-pure water to 50ul.
(2) PCR conditions：Pre-degeneration 95 DEG C 5 minutes, denaturation 95 DEG C 30 seconds, annealing 52~60 DEG C 30 seconds, extend 72 DEG C 2 points
Clock, is circulated 30 times, eventually extension 8 minutes.
The template DNA of 1st PCR derives from product phosphorus solar month of 30 days bacterium JN459, and the template DNA of the 2nd PCR and follow-up PCR uses
Last time PCR product.
The construction method of the strain storehouse containing ppk2 gene mutation promoters, comprises the following steps that：
(1) the shuttle vector pMV306K of structure carrying ppk2 gene upstream and downstream homology arms first, then by continuous error-prone
PCR product after Agar Gel sugar electrophoretic separation with PCR product is recycled, with above-mentioned shuttle vector pMV306K in Gibson
Cloning Master Mix effects are lower to carry out vitro recombination, converts escherichia coli DH5a, and picking transformant extraction plasmid carries out
Double digestion is identified；
(2) JN459 bacterial strains are transferred to by pMV306K plasmids are recombinated using electroporated technology, under homologous recombination effect
To a series of transformants, the strain storehouse of the mutant of gene promoter containing ppk2 is built.
Beneficial effects of the present invention：
(1) present invention screens the mutation of Polyphosphate kinase ppk2 gene promoters by the way of orthomutation first
Body, while obtain high-performance product phosphorus solar month of 30 days bacterium JN459- by detecting Poly-P changes of contents under aerobic and anaerobism service condition
M571, its anaerobic condition operation 24 it is small when after Poly-P contents than 2.4 times of wild-type bacteria plant height, can be applied to sewage treatment plant
EBPR Biological Phosphorus Removal Processes, have important economic value and social value.
(2) present invention is mutated the promoter sequence of Polyphosphate kinase ppk2 using continuous error-prone PCR method in vitro, subtracts
Weak activation of the hypoxia condition to ppk2 genes, reduces the expression of ppk2, reduces PPK2 and Poly-P is divided
Solution, ultimately improves the performance of long-pending phosphorus solar month of 30 days bacterium accumulation Poly-P.
(3) Poly-P decomposition rates are greatly lowered product phosphorus solar month of 30 days bacterium JN459-M571 of the invention under anaerobic,
And Poly-P performances are synthesized under aerobic condition without significant difference.
(4) preparation method of product phosphorus solar month of 30 days bacterium engineering bacteria of the invention, is mutated the efficient of work, and specific aim is very strong,
The randomness and blindness of mutation breeding are reduced, meets the breeding needs of microorganism fungus kind.
Brief description of the drawings
Fig. 1：To the removal effect of titanium pigment in sewage under aerobic and anaerobism service condition；
Fig. 2：Poly-P changes of contents under aerobic and anaerobism service condition.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, the description below is merely to explain this
Invention, is not defined its content.
Embodiment 1：Continuous error-prone PCR obtains the ppk2 promoters containing mutating alkali yl
The promoter of ppk2 genes is mutated in vitro using continuous error-prone PCR.Specially：
1. the primer of fallibility PCR is：
Sense primer ppk2ProForward sequences：GTCGAAATGTCGGAAGCCGTGC(SEQ ID NO.2)；
Anti-sense primer ppk2ProReverse sequences：TAACCCATTGTTCGCCCGGCAG(SEQ ID NO.3).
(1) PCR reaction systems：1~3ul of template DNA 2ul, PCR buffer solution 5ul, Taq archaeal dna polymerase 1ul, dATP,
1~3ul of dTTP, 1~3ul of dGTP, 1~3ul of dCTP, sense primer 2ul, anti-sense primer 2ul, 25mM MgCl21~8ul,
5mM MnCl20~1ul, adds ultra-pure water to 50ul.
(2) PCR conditions：Pre-degeneration 95 DEG C 5 minutes, denaturation 95 DEG C 30 seconds, annealing 52~60 DEG C 30 seconds, extend 72 DEG C 2 points
Clock, is circulated 30 times, eventually extension 8 minutes.
The template DNA of 1st PCR derives from product phosphorus solar month of 30 days bacterium JN459, and the template DNA of the 2nd PCR and follow-up PCR uses
Last time PCR product.
PCR product directly send sequencing, the results show：When dATP 3ul in PCR system, dTTP 3ul, dGTP 1ul,
DCTP1ul, sense primer 2ul, anti-sense primer 2ul, 25mM MgCl23ul、5mM MnCl20.5ul, PCR annealing temperature are 56
DEG C, base mismatch number is 3~6 during 3 consecutive PCRs, and the frequency of mutation is relatively appropriate for directional transformation.
Embodiment 2：The mutant library structure of product phosphorus solar month of 30 days bacterium ppk2 gene promoter
Structure carries the shuttle vector pMV306K of ppk2 gene upstream and downstream homology arms, by the continuous error-prone in embodiment 1
PCR product recycles PCR product after being separated with agarose gel electrophoresis, with above-mentioned shuttle vector pMV306K in Gibson
Cloning Master Mix effects are lower to carry out vitro recombination, converts escherichia coli DH5a, and picking transformant extraction plasmid carries out
Double digestion identifies (pMV306K is conventional plasmid of the prior art).
Fluid nutrient medium is prepared by following formula：Glucose 0.5g, peptone 0.5g, dusty yeast 0.5g, sodium glutamate
0.5g, ammonium sulfate 0.1g, dipotassium hydrogen phosphate 0.44g, magnesium sulfate 0.1g, purified water 1L, pH 7.0,0.15MPa sterilizing 30min.
Per the bottled 50ml fluid nutrient mediums of 250ml triangles.
By product phosphorus solar month of 30 days bacterium JN459 inoculations to aforesaid liquid culture medium, 25 DEG C, 200rpm shaking table cultures to logarithm life
For a long time, thalline is collected by centrifugation, is washed 3 times with 4 DEG C of 10% glycerine, is finally diluted to 10 with 4 DEG C of 10% glycerine8A/ml, as electricity
Hit the competent cell of conversion.
Long-pending phosphorus solar month of 30 days bacterium JN459 bacterial strains are transferred to by pMV306K plasmids are recombinated using electroporated technology, are made in homologous recombination
A series of transformants are obtained under, build the strain storehouse of the mutant of gene promoter containing ppk2, obtain 1035 recombinant bacteriums altogether
Strain, numbering JN459-M1 to JN459-M1035.
The gene order and gene location of above-mentioned ppk2 genes and its upstream and downstream are well known in the prior art, are documented in
Kawakoshi A.,Nakazawa H.,Fukada J.,Sasagawa M.,Katano Y.,Nakamura S.,Hosoyama
A.,Sasaki H.,Ichikawa N.,Hanada S.,Kamagata Y.,Nakamura K.,Yamazaki S.,Fujita
N.Deciphering the genome of polyphosphate accumulating Actinobacterium
Microlunatus phosphovorus.DNA Research.2012,19:383-394.Recombinant shuttle vector pMV306K's
The method that structure uses is that general technique for gene engineering is (such as big with reference to " Principles of Gene Engineering and technology ", Zou Keqin, Zhejiang
Learn publishing house), for the conventional technical means of the prior art.
Product phosphorus solar month of 30 days bacterium JN459 bacterial strain is documented in document " separation of polyP bacteria JN459 and poly- phosphorus characteristic research ", Zhong Chuan
Green grass or young crops, Jiang Tianyi, Wang Jing, Zhang Chunming Journal of Shandong Jianzhu University .201530 (1):In 25-28.
Embodiment 3：The poly- phosphorus screening active ingredients of mutant strain
The recombinant bacterial strain that embodiment 2 is built is cultivated to exponential phase, centrifugation with above-mentioned fluid nutrient medium (embodiment 2)
Collect thalline, and with sterile water washing, be diluted to 108A/ml, new aforesaid liquid culture medium is inoculated into by 5% inoculum concentration, often
A recombinant bacterial strain is inoculated with a 50ml/250ml shaking flask.25 DEG C, 200rpm shaking table cultures 48 it is small when, draw 10ml bacterium solutions,
3000rpm centrifugations 10min collects thalline, measures Poly-P contents.Remaining nutrient solution replaces air in shaking flask with sterile nitrogen,
Then seal bottleneck, 25 DEG C, 200rpm shaking tables continue culture 24 it is small when, draw 10ml bacterium solutions, 3000rpm centrifugations 10min is collected
Thalline, measures Poly-P contents.It the results are shown in Table 1.
17 plant mutant strain of the results show when anaerobism 24 is small after Poly-P contents be significantly higher than starting strain JN459, wherein
The Poly-P content highests of JN459-M571 mutant bacterias, reach 8.62%, illustrate that these mutant strains decompose under anaerobic
The ability of Poly-P declines.
Table 1 is aerobic and Poly-P accounts for dry cell weight (%) during anaerobism operation
Embodiment 4：The genetic stability of product phosphorus solar month of 30 days bacterium JN459-M571 bacterial strain is investigated
Solid medium is prepared by following formula：Glucose 0.5g, peptone 0.5g, dusty yeast 0.5g, sodium glutamate
0.5g, ammonium sulfate 0.1g, dipotassium hydrogen phosphate 0.44g, magnesium sulfate 0.1g, agar powder 20g, purified water 1L, pH 7.0,0.15MPa
Sterilize 30min.
JN459-M571 strain suspensions dilution spreads are cultivated 7 days for 25 DEG C on solid medium, then separate JN459-
The single bacterium colony of M571 is simultaneously enlarged culture, continuous passage culture and separation monospore six times, and according to embodiment 3 in this way
The poly- phosphorus activity of method validation, the results are shown in Table 2, the results showed that JN459-M571 bacterial strains have preferable genetic stability, meet
The demand of EBPR Biological Phosphorus Removal Systems.
The poly- phosphorus expression activitiy of the different passage numbers of table 2
Embodiment 5：The determined dna sequence of ppk2 gene promoter mutant
The genomic DNA for extracting engineering bacteria JN459-M571 is used as template, with ppk2ProForward with
Ppk2ProReverse carries out high-fidelity PCR amplification for primer, and PCR product is separated and recovered from through agarose gel electrophoresis, with
PCR-Blunt carriers connection conversion escherichia coli DH5a, the several positive colonies of picking carry out DNA sequencing.In JN459-M571
The DNA sequence dna of ppk2 promoters is as shown in SEQ ID NO.1 in sequence table, the DNA sequence dna of ppk2 promoters such as sequence in JN459
In table shown in SEQ ID NO.4.The results show that ppk2 gene promoters have the change of 3 bases in JN459-M571, respectively
It is that -46, -76 and -79 bit bases become A by G.
It is common that bacterial strain JN459-M571 on December 1st, 2015 is preserved in China Committee for Culture Collection of Microorganisms
(abbreviation CGMCC, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research at microorganism center
Institute), deposit number is CGMCC NO.11770.
Embodiment 6：JN459-M571 verifies the removal effect of titanium pigment in sewage
Artificial synthesized sewage is prepared by following ingredients：Glucose 0.3g, peptone 0.1g, dusty yeast 0.01g, sodium acetate
0.15g, ammonium chloride 0.2g, sodium chloride 0.05g, dipotassium hydrogen phosphate 0.1g, magnesium sulfate 0.12g, purified water 1L.Survey NH4 +- N is
65.1mg/L PO4 3-- P is 14.2mg/L.
By JN459-M571 strain liquids culture to exponential phase, thalline is collected by centrifugation, and with sterile water washing, then
It is inoculated into 10L sbr reactor devices.Using aerobic and Anoxic technique, aerobic time is 12h in each cyclic process, dissolving
Oxygen concentration is about 4mg/L, hypoxic exposure 12h, replaces the hollow gas of system with sterile nitrogen, maintains dissolved oxygen concentration to be less than
0.2mg/L, whole technique maintain about 3d, and sampling detects titanium pigment content in water phase, and the results are shown in Figure 1；Run later stage inspection
Poly-P contents in thalline are surveyed, using JN459 bacterial strains as control, the results are shown in Figure 2.Operation result shows, JN459-M571 bacterium
Strain titanium pigment ascensional range when anaerobism is run between 7.7%-10.1%, is substantially less than JN459 bacterial strains (23.9%-
Between 31.3%).Titanium pigment removal rate of the JN459-M571 bacterial strains when 72 is small reaches 79.8% at the same time, is significantly higher than
59.8% removal rate of JN459 bacterial strains；Intracellular Poly-P contents reach the 8.6% of dry cell weight, also above N459 bacterial strains
4.3%.
Claims (5)
1. the ppk2 gene promoters of nucleotide sequence such as SEQ ID NO.1 a kind of reduce ppk2 expression water under low oxygen conditions
Flat application.
2. the product phosphorus solar month of 30 days bacterium engineering bacteria of one plant of efficient accumulation polyphosphate, which is product phosphorus solar month of 30 days bacterium
（Microlunatus phosphovorus）JN459-M571, has been preserved in Chinese microorganism strain guarantor on December 1st, 2015
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC NO.11770.
3. a kind of microbial inoculum, it is characterised in that the active ingredient of the microbial inoculum is the product phosphorus solar month of 30 days bacterium described in claim 2
（Microlunatus phosphovorus）JN459-M571.
4. product phosphorus solar month of 30 days bacterium described in claim 2（Microlunatus phosphovorus）JN459-M571 and/or right will
Ask application of 3 microbial inoculums in biological phosphate-eliminating agent is prepared.
5. the primer of the fallibility PCR for directional transformation ppk2 gene promoters, it is characterised in that its primer sequence is respectively such as
Shown in SEQ ID NO.2 and SEQ ID NO.3.
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| CN106479928B (en) * | 2016-11-02 | 2019-10-11 | 中国科学院微生物研究所 | The indigenous plasmid of one plant of resistance to resistance to high COD salt water meningitidis strains and the source bacterial strain with high salt |
| CN107162188B (en) * | 2017-05-18 | 2020-07-03 | 北京工业大学 | Device and method for integrated autotrophic nitrogen removal and synchronous reinforcement of biological phosphorus removal |
| CN110904069A (en) * | 2019-12-04 | 2020-03-24 | 天津市职业大学 | PPK2 protein and application thereof in polyacrylamide gel electrophoresis 35kd standard substance |
| CN111170471B (en) * | 2020-01-16 | 2021-12-17 | 浙江永续环境工程有限公司 | Composite phosphorus-accumulating bacteria flowing biological bed membrane method |
| CN112322664B (en) * | 2020-11-02 | 2021-07-27 | 江苏南创化学与生命健康研究院有限公司 | Method for producing polyphosphate with high polymerization degree |
| CN112779021B (en) * | 2021-02-03 | 2021-10-15 | 中南大学 | Phosphorus-containing heavy metal contaminated soil remediation material and preparation method and application thereof |
| CN114561560A (en) * | 2022-03-02 | 2022-05-31 | 广西惟邦环境科技有限公司 | Dephosphorization treatment method for extracting high-grade and low-grade manganese ores |
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