CN104531599A - Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof - Google Patents

Abstract

The invention discloses a strain of Citrobacter freundii transmuted with a phosphorus-accumulating gene, which is a Citrobacter freundii introduced with its own polyphosphate kinase Ppk1 gene. There is only the polyphosphate kinase gene Ppk1 in the Citrobacter freundii genome DNA, and the regulation mode of the Ppk1 gene and the exopolyphosphatase gene Ppx is bicis-trans co-transcription. Bacteria with the above characteristics can improve their ability to accumulate phosphorus by expressing their own Ppk1 gene with the host itself as a receptor. The invention also discloses the construction method of the above-mentioned Citrobacter freundii transgenic gene and its application in wastewater phosphorus removal. The phosphorus-accumulating gene-transferred Citrobacter freundii obtained by the invention has the characteristics of strong dephosphorization ability.

Description

A strain of Citrobacter freundii transgenic for phosphorus accumulation gene and its construction method and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a strain of Citrobacter freundii transmuted with a phosphorus-accumulating gene and its construction method and application.

Background technique

In freshwater bodies, controlling or alleviating eutrophication largely depends on controlling the input of phosphorus nutrients. The sources of phosphorus-containing nutrients input into water bodies are various, including natural processes and human activities, but the main reason that causes excessive phosphorus in water bodies and leads to eutrophication of water bodies is mainly human activities. In my country, the main sources of phosphorus-containing pollutants are domestic pollution sources, agricultural pollution sources, livestock and poultry pollution sources, and industrial pollution sources. In order to solve the harm caused by phosphorus pollution, it is necessary to effectively treat the sewage before it enters the water body and reduce the phosphorus concentration in the discharged sewage.

At present, there are two main types of phosphorus removal technologies in sewage, namely chemical phosphorus removal and biological phosphorus removal. In the chemical phosphorus removal technology, the chemical precipitation method is the most used. The basic idea is to add cheap chemical precipitants in different links of the sewage treatment process. These precipitants generally have a flocculation function. The precipitants and sewage Phosphate reacts to coagulate into granular or insoluble flocculated precipitates, thereby removing soluble phosphorus. Chemical phosphorus removal has the advantages of simple operation, high removal rate, and stable operation. However, for urban sewage with low phosphorus concentration, it is necessary to separate low-concentration phosphate from aqueous solution in the form of chemical precipitation, and excessive chemical Precipitating agent is the basic condition to ensure the quality of effluent water. Therefore, this method has disadvantages such as large dosage, high treatment cost, large amount of chemical sludge and rich in organic matter, etc., which is not conducive to its application in the actual sewage treatment process. Biological phosphorus removal technology is mainly derived from the excessive phosphorus uptake phenomenon observed by Srinath et al. in production and operation in the late 1950s and early 1960s. After years of systematic and comprehensive basic research, productive research and engineering operation summary, several mainstreams have been formed. Phosphorus removal processes mainly include A/O process, Phoredox (Bardenpho) process, oxidation ditch process, and sequencing batch activated sludge process (SBR), etc., no matter which of the above processes utilizes the principle of biological overaccumulation of phosphorus . There are two main methods for the biological removal of phosphorus in sewage: one is that the phosphorus required for the normal growth of microorganisms is removed with the elimination of organisms; the other is that the phosphorus accumulating organisms (Phosphorus accumulating organisms , PAOs) decompose polyphosphate (Polyphosphate, Polyp) under anaerobic conditions while aggregating poly-β-hydroxybutyric acid (Poly-β-hydroxybutyric acid, PHB), decompose PHB under aerobic conditions to obtain energy for Phosphate is actively absorbed and stored in the body in the form of Polyp, which is removed as it is eliminated by the organism. At present, the phosphorus removal methods commonly used in large-scale sewage treatment plants around the world are mainly biological phosphorus removal, especially the enhanced biological phosphorus removal method (EBPR), because of its advantages of less sludge generation, no use of chemicals and operation economic characteristics.

Polyp is a linear polymer formed by more than 3-1000 orthophosphate groups ranging from high-energy phosphate bonds similar to those in adenosine triphosphate (ATP), which widely exists in bacteria , fungi and other lower single-celled organisms and higher mammalian cells. Although the exact physiological function of this compound full of high-energy phosphate bonds has not been fully understood so far, it is certain that it is closely related to a variety of biological functions, including: (1) phosphate and high-energy Storage of phosphate bonds; (2) Chelating agent for divalent cations; (3) Interaction with ribosomal proteins to promote the translation process, and may be related to the correction of mistranslations; (4) Promote stringent response (Stringent response), etc.;

As far as we know, multiple metabolic pathways are related to the synthesis of Polyp, and the most widely accepted view is that the synthesis of Polyp in most microorganisms is catalyzed by the polyphosphate kinase (Ppk) encoded by their genomes. Ppk includes two families, Ppk1 and Ppk2, which each catalyze the following two reversible reactions:

In addition to different substrate preferences, the most significant difference between Ppk1 and Ppk2 is that Ppk1 tends to synthesize Polyp, while Ppk2 tends to hydrolyze Polyp.

Since Ppk (belonging to the Ppk1 family) was first discovered in Escherichia coli (Escherichia coli, E.coli), its related coding gene has been cloned, and the Ppk1 protein translated into it has been purified. Genetic methods are used to achieve bioaugmented phosphorus removal. At present, there have been reports that the Ppk1 gene of E.coli has been expressed in E.coli and achieved significant results. However, in view of the particularity of E.coli's identity, its environmental application remains to be discussed.

Contents of the invention

The technical problem to be solved by the present invention is to provide a strain of Citrobacter freundii with a phosphorus-accumulating gene, which can efficiently remove phosphorus in wastewater.

The technical problem to be solved by the present invention is to provide a construction method of the above-mentioned Citrobacter freundii transgenic for phosphorus accumulation and its application in wastewater phosphorus removal.

The final technical problem to be solved by the present invention is to provide a bacterial biomodification strategy and method for improving the phosphorus accumulation ability of bacteria having similar phosphorus accumulation gene characteristics with Citrobacter freundii.

In order to solve the problems of the technologies described above, the present invention adopts the following technical methods:

A strain of Citrobacter freundii with polyphosphorus gene transfection takes Citrobacter freundii as the host and introduces the host's own polyphosphate kinase Ppk1.

Wherein, there is only Ppk1 polyphosphate kinase gene in the genomic DNA of Citrobacter freundii, and the regulation mode of the Ppk1 gene and the exopolyphosphatase gene Ppx on the genome is bicistronic co-transcription .

Wherein, the Citrobacter freundii is Citrobacter freundii ATCC 8090, and the GenBank number of the polyphosphate kinase Ppk1 gene is ANAV01000007.1.

The construction method of the above-mentioned Citrobacter freundii of transgenic phosphorus accumulation gene comprises the following steps:

(1) Acquisition of Citrobacter freundii polyphosphate kinase Ppk1 gene:

According to the Citrobacter freundii ATCC 8090 (Citrobacter freundii ATCC8090) "whole genome shotgun sequence" provided in the NCBI database, design two primers, the primer sequence is as follows: SEQ ID NO.3: Forward primer (named as CFPPKF): 5 '-GGGGTACCAatgggtcaggaaaagctatacatcg-3', SEQ ID NO.4: Reverse primer (designated as CFPPKR): 5'-CCCAAGCTTttagtcaggttgctcgagtgatttg-3',

Using Citrobacter freundii ATCC 8090 genomic DNA as a template, carry out PCR amplification, the amplification conditions are: 95°C for 5min, 98°C for 10Sec, 68°C for 2min, 30 cycles, 72°C for 5min, to obtain the Ppk1 gene fragment;

(2) Construction of T-Ppk1 plasmid:

The Ppk1 gene fragment obtained in step (1) was recovered using a PCR product recovery kit, and the Ppk1 gene fragment was subjected to an A reaction, and after recovery again, it was connected to the T-Vector pMD19 (Simple) vector with T4 ligase to obtain T-Vector pMD19 (Simple) - Ppk1 plasmid, transform it into E.coli DH5α competent cells by heat shock, screen on the LB plate containing 100μg/ml Amp, extract the plasmid and verify it, then send it to the sequencing company to determine the sequence of the insert fragment, and use Bio-Edit software to perform Sequence analysis;

(3) Construction of pBBR1MCS-Ppk1 plasmid:

Use restriction endonucleases Kpn I and Hind III to digest the T-Ppk1 plasmid, and double-enzyme-cut the sequenced Citrobacter freundii Ppk1 gene fragment from the T-Vector pMD19 (Simple) vector, and use gel recovery The kit recovers the target fragment, and uses T4 ligase to connect the target fragment with the broad-host expression vector pBBR1MCS-2 that has undergone the same double digestion and recovery to obtain the pBBR1MCS-Ppk1 plasmid, which is transformed into E.coliDH5α by heat shock State cells were screened on an LB plate containing 50 μg/ml Kana, the extracted plasmid was double-enzymatically digested and verified, and then submitted to the sequencing company to determine the sequence of the insert fragment, and sequence analysis was performed with Bio-Edit software to ensure that the sequence was correct.

(4) Preparation of Citrobacter freundii electrotransformation competent cells:

The wild-type strain of Citrobacter freundii ATCC8090 (named: CF-WT) was streaked on an LB plate, and cultured statically at 37° C. for 12 hours to obtain a single colony. Pick a single colony of CF-WT in 3ml LB medium, culture at 37°C with shaking at 200rmp for 12hr. According to the inoculation ratio of 1:100 (V/V), CF-WT was inoculated in 50ml LB medium, 37°C, 200rmp shaking culture to OD600=0.35. The obtained culture was placed in an ice bath for 30 min, with gentle shaking from time to time to ensure sufficient cooling of the culture. At 4°C and 2500 rpm, centrifuge for 10 min and discard the supernatant to collect the cells. Resuspend the cells in 30ml ice-bath pre-cooled sterile deionized water, centrifuge at 2500rpm for 10min at 4°C, and discard the supernatant. Repeat the suspension once more and discard the supernatant. The bacterial cells were resuspended with 1ml of 10% (V/V) glycerol pre-cooled in an ice bath, and distributed into 100μl/1.5ml centrifuge tubes, and placed on ice for later use.

(5) Construction of the Citrobacter freundii of the phosphorus accumulation gene:

Using the method of electroporation, the constructed broad-host expression vector pBBR1MCS-2 containing the Citrobacter freundii Ppk1 gene was transformed into Citrobacter freundii ATCC 8090, and screened on an LB plate containing 50 μg/ml Kana. The strain from which the expression vector is extracted is the Citrobacter freundii transmuted with the phosphorus accumulation gene.

The application of the above-mentioned Citrobacter freundii transgenic for phosphorus accumulation gene in phosphorus removal from wastewater is also within the protection scope of the present invention.

Wherein, the said wastewater has a phosphorus content of 0.1-20 mg/L.

A method for improving the ability of bacterial strains to accumulate phosphorus in wastewater. The method uses a bacterial strain with only Ppk1, a polyphosphate kinase gene, as a host bacterium, and the Ppk1 gene in the host bacterium is combined with exopolyphosphate The regulatory mode of the enzyme gene Ppx is bicistronic co-transcription, the Ppk1 gene of the host bacterium is obtained by PCR, and constructed into an expression plasmid, and finally the expression plasmid connected with the Ppk1 gene is transformed into the host bacterium, and the transgenic gene is obtained. The host bacterium of the phosphorus-accumulating gene, the host bacterium is Acinetobacter baumannii, Clostridium acetobutylicum, Rhodospirillum, Nitrosomonas, Serratia or Shewanella.

Beneficial effect:

1. The present invention obtains the Ppk1 gene derived from Citrobacter freundii ATCC 8090 by PCR, and constructs the gene fragment into the broad host expression vector pBBR1MCS-2 by restriction enzyme digestion to obtain the pBBR1MCS-Ppk1 plasmid, and then transforms the pBBR1MCS-Ppk1 plasmid Citrobacter freundii ATCC 8090 obtained Citrobacter freundii transgenic for phosphorus accumulation.

2, the maximum OD value of the Citrobacter freundii of the transphosphorus-accumulating gene that the present invention obtains reaches about 0.58 in phosphorus-containing wastewater, but the maximum OD value of the contrast bacterial strain that does not transpose the phosphorus-accumulating gene can only reach about 0.47, so , transfection of phosphorus-accumulating gene is beneficial for Citrobacter freundii to survive and accumulate phosphorus in phosphorus-containing wastewater, thereby effectively removing phosphorus in wastewater.

3. The phosphorus-accumulating gene-transferred Citrobacter freundii constructed by the present invention can remove phosphorus in wastewater by 35% compared with the reported Escherichia coli BL-PPK strain.

Description of drawings

Fig. 1 Agarose gel electrophoresis image of PCR product of Citrobacter freundii Ppk1 gene.

Fig. 2 The agarose gel electrophoresis picture of the pBBR1MCS-2 empty vector and the connection with Citrobacter freundii Ppk1.

Fig. 3 The growth curve of biomodified Citrobacter freundii in synthetic wastewater.

Fig. 4 The change of phosphorus concentration in synthetic wastewater by adding biologically modified Citrobacter freundii.

Fig. 5 Staining pictures of intracellular heterochromas of biologically modified Citrobacter freundii (CF-MPPK).

Fig. 6 Structural diagram of Ppx and Ppk1 in the genomic DNA of Pseudomonas putida KT2440.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

The strains and plasmid sources used in the following examples are as follows:

(1) Escherichia coli E.coli DH5α is preserved by our laboratory;

(2) Citrobacter freundii (Citrobacter freundii) was purchased from China Center of Industrial Culture Collection (CICC), strain preservation number 10296, and its ATCC preservation number 8090 (American Type Culture Collection, ATCC, American Type Culture Collection);

(3) Plasmid T-Vector pMD19 (Simple): purchased from Treasure Bioengineering (Dalian) Co., Ltd. (TARAKA);

(4) Plasmid pBBR1MCS-2: a broad host expression vector, preserved by our laboratory.

The sources of enzymes and other reagents used in the following examples are as follows:

Restriction endonuclease, ligase, Taq DNA polymerase, Prime STAR DNA polymerase, and four dNTP master mixes were purchased from TARAKA Bioengineering (Dalian) Co., Ltd. (TARAKA);

Tryptone and Yeast extract were purchased from British OXOID company;

Antibiotics were purchased from Shanghai Sangon Bioengineering Co., Ltd.;

The rest of the conventional chemical reagents were of domestic analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.;

PCR product purification kit, DNA gel recovery kit, and plasmid mini-extraction kit were purchased from Corning Life Sciences (Wujiang) Co., Ltd.;

The bacterial genome DNA extraction kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Example 1: Construction of Citrobacter freundii transgenic for phosphorus accumulation gene.

(1) PCR amplification of Citrobacter freundii Ppk1 gene:

According to the Citrobacter freundii ATCC 8090 "whole genome shotgunsequence" provided in the NCBI database, GenBank accession number: ANAV01000007.1, design two primers-forward primer (named CFPPKF): 5'-GGGGTACCAatgggtcaggaaaagctatacatcg-3', reverse primer ( Named as CFPPKR): 5'-CCCAAGCTTttagtcaggttgctcgagtgatttg-3', using Citrobacter freundii ATCC 8090 genomic DNA as a template, PCR amplification, amplification conditions: 95°C for 5min, 98°C for 10Sec, 68°C for 2min, 30 cycles, 72 ℃ 5min.

(2) Sequence analysis of Citrobacter freundii Ppk1 gene:

The PCR product was cleaned and recovered for adding A reaction, and then cleaned and recovered again and connected to the T-Vector pMD19 (Simple) vector to obtain the T-Ppk1 plasmid, which was transformed into E.coli DH5α competent cells by heat shock, in a medium containing 100 μg/ml Screening was carried out on the LB plate of Amp, and after the plasmid was extracted and verified, the sequence of the insert was determined by Nanjing Sipujin Biotechnology Co., Ltd., and the sequence was analyzed by using Bio-Edit software.

(3) Construction of expression vector:

Use restriction endonucleases Kpn I and Hind III to double-enzyme excise the Citrobacter freundii Ppk1 gene fragment sequenced correctly from the T-Vector pMD19 (Simple) carrier, use the gel recovery kit to recover the target fragment, and use the target The fragment was connected with the broad host expression vector pBBR1MCS-2 that had been digested and recovered by the same double restriction enzymes to obtain the pBBR1MCS-Ppk1 plasmid, which was then transformed into E.coli DH5α competent cells by heat shock, and carried out on the LB plate containing 50 μg/ml Kana After screening, extracting the plasmid and verifying it by double enzyme digestion, it was sent to Nanjing Sipujin Biotechnology Co., Ltd. to determine the sequence of the insert fragment, and the sequence analysis was performed with Bio-Edit software to ensure that the sequence was correct.

(4) Preparation of Citrobacter freundii electrotransformation competent cells:

The wild-type strain of Citrobacter freundii (named: CF-WT) was streaked on an LB plate, and cultured at 37°C for 12 hours to obtain a single colony. Pick a single colony of CF-WT in 3ml LB medium, culture at 37°C with shaking at 200rmp for 12hr. According to the inoculation ratio of 1:100 (V/V), CF-WT was inoculated in 50ml LB medium, 37°C, 200rmp shaking culture to OD600=0.35. The obtained culture was placed in an ice bath for 30 min, with gentle shaking from time to time to ensure sufficient cooling of the culture. At 4°C and 2500 rpm, centrifuge for 10 min and discard the supernatant to collect the cells. Resuspend the cells in 30ml ice-bath pre-cooled sterile deionized water, centrifuge at 2500rpm for 10min at 4°C, and discard the supernatant. Repeat the suspension once more and discard the supernatant. Resuspend the bacteria in 1ml of 10% (V/V) glycerol pre-cooled in an ice bath, aliquot into 100μl/1.5ml centrifuge tubes, and place on ice for later use.

(5) Construction of transgenic Citrobacter freundii and its control strains:

Using the method of electroporation, the broad-host expression vector pBBR1MCS-2 containing Citrobacter freundii Ppk1 gene constructed in step (3) was transferred into Citrobacter freundii ATCC 8090, in LB containing 50 μg/ml Kana Screening is carried out on the plate, and the bacterial strain from which the expression vector can be extracted is the transgenic Citrobacter freundii (named as CF-MPPK).

Using the same method, transfer the empty-loaded broad-host expression vector pBBR1MCS-2 into Citrobacter freundii ATCC8090, screen on the LB plate containing 50 μg/ml Kana, and extract the strain of the empty-loaded expression vector It is Citrobacter freundii (named: CF-M) used for comparison.

Example 2: PCR amplification results of Citrobacter freundii Ppk1 gene.

According to the Citrobacter freundii ATCC 8090 (whole genome shotgunsequence) GenBank accession number provided in the NCBI database: ANAV01000007.1, the designed primers amplified a Ppk1 gene fragment of 2067 bp in size from the Genomic DNA of Citrobacter freundii ATCC8090 (detailed sequence See the sequence table (SEQID NO.1), as shown in Figure 1, the position of the band is correct, and the sequence result is compared with the sequence reported in the NCBI database using Bio-Edit software, and the base sequence is also completely consistent.

Example 3: Construction results of Citrobacter freundii Ppk1 gene expression vector.

The full length of the broad host expression vector pBBR1MCS-2 is 5144bp (see the sequence table SEQ ID NO.2 for detailed sequence), as shown in Figure 2, after inserting the Ppk1 gene fragment of Citrobacter freundii 2067bp in the multiple cloning site of this expression vector, The vector became significantly larger, and the sequence was completely correct after double enzyme digestion and sequencing.

Example 4: Growth of CF-MPPK strain in synthetic wastewater and determination of phosphorus removal effect.

(1) Streak CF-WT on LB plates to obtain single colonies, respectively streak CF-MPPK and CF-M on LB plates containing 50 μg/ml Kana, and culture at 37°C for 12 hours to obtain single colonies .

(2) Pick CF-WT single colonies in 3ml LB medium, 37°C, 200rpm shaking culture for 12hr; pick CF-MPPK and CF-M single colonies respectively in 3mlLB medium containing 50μg/ml Kana, 37°C, 200rpm Incubate with shaking for 12hr.

(3) According to the inoculation ratio of 1:100 (V/V), inoculate CF-WT in 20ml LB medium, 37 ° C, 200rpm shaking culture for 12hr; according to the inoculation ratio of 1:100 (V/V), respectively CF-MPPK and CF-M were inoculated in 20ml LB medium containing 50μg/ml Kana, 37°C, 200rpm shaking culture for 12hr.

(4) Measure the OD600 values of CF-WT, CF-MPPK, and CF-M after the cultivation respectively, and take an appropriate volume of bacterial liquid (the volume is converted according to the OD600 value in the LB medium, so that the initial OD600 in the synthetic wastewater after inoculation value is around 0.2), respectively in 10ml sterile centrifuge tubes, room temperature, 5000rpm centrifugation for 5min to collect bacteria, after discarding the supernatant, resuspend the bacteria in 5ml of sterile deionized water, room temperature, 5000rpm centrifugation for 5min to collect bacteria , discard the supernatant and wash the cells again in the same way.

(5) Resuspend the bacteria with 1ml of synthetic wastewater medium, inoculate CF-WT into 100ml of synthetic wastewater medium, culture at 37°C and 200rpm for 12hrs, and inoculate CF-MPPK and CF-M respectively in the same way In the synthetic wastewater medium containing 50μg/ml Kana, 37°C, 200rpm shaking culture for 12hr, during which the culture solution of the three strains was sampled every two hours, and the OD600 value of each strain was measured respectively, and the synthetic wastewater Phosphorus content in the serum.

Example 5: Biomodification of the growth curve of Citrobacter freundii in synthetic wastewater.

The formula of synthetic wastewater is as follows: glucose 0.3g, Tryptone 0.1g, Yeast extract 0.01g, anhydrous sodium acetate 0.15g, sodium chloride 0.05g, magnesium sulfate heptahydrate 0.262g, dipotassium hydrogen phosphate trihydrate 1.472g, chloride Ammonium 0.18g, deionized water 1L, sterilized at 115°C for 30min for later use, the total phosphorus content is 20mg/L.

It can be seen from Figure 3 that when the strains grow in the synthetic wastewater medium with an initial OD value of about 0.20, the growth range of Citrobacter freundii ATCC 8090 (CF-WT) and the strain containing the empty carrier (CF-M) are relatively close. The maximum OD value can only reach about 0.47, while the growth rate of the strain expressing the Citrobacter freundii Ppk1 gene, namely CF-MPPK, is significantly greater than the former two, with a maximum OD value of about 0.58, which is consistent with the previous reports of the decrease in exogenous nutrients. When , polyphosphate can promote the growth of Escherichia coli with similar results. Facilitating phosphorus accumulation by Citrobacter freundii by expressing the Citrobacter freundii Ppk1 gene.

Example 6: Phosphorus removal effect of biologically modified Citrobacter freundii in synthetic wastewater.

For the determination method of total phosphorus in synthetic wastewater, refer to "National Standard of the People's Republic of China GB11893-89".

It can be seen from Figure 4 that when each strain grows in synthetic wastewater with a phosphorus content of 20 mg/L, the phosphorus removal effect of Citrobacter freundii ATCC 8090 (CF-WT) and the strain containing the empty carrier (CF-M) is much lower than that of the expression The strain CF-MPPK carrying the Citrobacterfreundii Ppk1 gene, after 12 hours of cultivation, the phosphorus content in the supernatant of the former two fluctuated slightly around 19 mg/L, while the phosphorus content in the supernatant of the latter had dropped to about 3 mg/L.

Example 7: Staining of intracellular phosphorus-accumulating granules of the Citrobacter freundii strain transfected with the phosphorus-accumulating gene.

Polyp's modified Albert staining method: make slides according to the conventional method; stain with A solution for 5 minutes, pour off A solution, wash once with water, and air-dry; stain with B solution for 1 minute, wash once with water, and air-dry; It is blue-black, and other parts of the bacteria are green.

At the 10th hour, take 0.5ml each of CF-WT, CF-MPPK and CF-M cultured in synthetic wastewater into 1.5ml centrifuge tubes, centrifuge at 12000rpm for 2min, discard the supernatant, and resuspend in 1ml deionized water Centrifuge the cells at 12000rpm for 2min, discard the supernatant, wash the cells once again in the same way, and finally resuspend the cells in 0.1ml of deionized water, and perform heterochromatic granules (Polyp particles) after making slices according to the conventional method After staining, observe the cell morphology and intracellular heterochromatic granules with an oil microscope.

It can be seen from Figure 5 that after the staining of Citrobacter freundii ATCC 8090 (CF-WT) and the strain containing the empty vector (CF-M) by the modified Albert method, there were no blue-black heterochromatic particles in the cells, only green However, the strain expressing the Citrobacter freundii Ppk1 gene, namely CF-MPPK, has obvious heterochromatic particles, namely Polyp particles, in the cell, especially at both ends, which is also consistent with the phosphorus removal of the strain in synthetic wastewater.

Example 8: Comparison of phosphorus accumulation ability of different phosphorus accumulating bacteria.

OD600 refers to the absorbance value of a certain solution at a wavelength of 600nm. One of its important applications is to use the absorbance of bacteria to measure the concentration of bacterial culture solution. The OD600 value is usually positively correlated with the biomass of bacteria. When evaluating the phosphorus-accumulating ability of a bacterium, in addition to the apparent indirect indicator of the reduction of phosphorus content in the phosphorus-containing culture system, the content of Polyp in the cells of the phosphorus-accumulating bacteria should also be included in the evaluation of the phosphorus-accumulating ability system, and should be the most important indicator, because it most directly reflects the phosphorus accumulation ability of phosphorus accumulating bacteria. Therefore, in order to reflect the phosphorus accumulation ability of a phosphorus accumulating bacteria relatively accurately, the phosphorus reduction in the medium is used as the "numerator", and the biomass of phosphorus accumulating bacteria, that is, OD600, is the "denominator". Indicating that the ability of the strain to accumulate phosphorus is stronger. According to the application number 201210033343.9 and the application number 200610038917.6, two Chinese patents have constructed two strains of BL-PPK and KT4PPK respectively, and compared the phosphorus accumulation ability of the above two strains with the CF-MPPK bacterial strain constructed by the present invention (such as shown in Table 1). Since the Ppk1 gene was first discovered in Escherichia coli, the use of Escherichia coli to express the Ppk1 gene and use it in wastewater phosphorus removal has achieved remarkable results, and the BL-PPK strain is currently known to have the best phosphorus accumulation effect. It can be seen from Table 1 that the phosphorus accumulation ability of the CF-MPPK strain constructed by the present invention is increased by about 35% compared with the BL-PPK strain.

Table 1 Comparison of phosphorus accumulation ability of different phosphorus accumulating bacteria

According to literature reports, according to the distribution of the Ppk gene in the genomic DNA, bacteria can be divided into three categories: (1) only the Ppk1 gene exists in the genomic DNA, such as Escherichia coli, Clostridium acetobutylicum, Neisseriameningitidis, etc.; (2) only the Ppk1 gene exists in the genomic DNA Ppk2 gene, such as Bacillus thuringiensis, Kineococcus radiotolerans, Nitrospira multiformis, etc.; (3) Both Ppk1 and Ppk2 genes exist in genomic DNA, such as Pseudomonas putida, Myxococcus xanthus, Frankia alni, etc. Escherichia coli DH5α, which uses a similar method and is reported to have a significant phosphorus accumulation effect, belongs to the first category, and Enterobacter cloacae, which is very close to Escherichia coli in bacteriological classification, has been confirmed by experiments. The method failed to make it have a significant phosphorus accumulation effect. Through the analysis of the complete genome DNA sequence in the NCBI database, it was found that both Ppk1 and Ppk2 genes existed in the Enterobactercloacae genome DNA. The same method was used to transform the model species of Pseudomonasputida (Pseudomonasputida) KT2440, but it failed to have a significant phosphorus accumulation effect, and KT2240 is a representative strain with both Ppk1 and Ppk2 genes in the genomic DNA. Therefore, the only Ppk1 gene in the host bacteria is the prerequisite for whether the biomodified bacteria have the ability to accumulate phosphorus.

E.coli DH5α strains expressing Citrobacter freundii Ppk1 gene, Enterobacter cloacae Ppk1 gene and Pseudomonas putida KT2440Ppk1 gene were respectively subjected to the same synthetic wastewater phosphorus removal test, and no obvious phosphorus accumulation effect was found. Although the Ppk gene exists widely in bacteria, and there are also conserved regions in its gene sequence, there are still certain differences between different species. The amino acid sequence homology between Citrobacter freundii, Enterobacter cloacae, Pseudomonas putida KT2440 and E.coli DH5αPpk1 polyphosphokinase is 96%, 94% and 34%, respectively, even the highly homologous Ppk1 is significantly expressed in different hosts Therefore, the host bacteria have a strong ability to recognize and adapt to their own Ppk1 gene. When constructing phosphorus-accumulating strains, the Ppk1 gene derived from the host itself will be more conducive to the host's phosphorus accumulation.

The regulation mode of exopolyphosphatase gene (Exopolyphosphatase, Ppx) and Ppk1 gene in the genome DNA of host bacteria is bicistronic co-transcription (Co-transcribed bicistronic). According to the existing literature and the genomic DNA sequence analysis provided in the NCBI database, the Ppx and Ppk1 genes of Escherichia coli and Citrobacter freundii form a Co-transcribed bicistronic structure in their genomic DNA, which is significantly different from the simultaneous presence in genomic DNA The type strain Pseudomonas putida KT2440 of Ppk1 and Ppk2, as shown in Figure 6, Ppx of KT2440 is closely adjacent to the Ppk1 gene and overlaps at its 3' part. This may also be one of the reasons why Pseudomonasputida KT2440 failed to obtain obvious phosphorus accumulation effect when expressing its own Ppk1 gene. Therefore, the regulation mode of exopolyphosphatase gene (Exopolyphosphatase, Ppx) and Ppk1 gene in the host bacterial genome DNA is bicistronic co-transcription (Co-transcribed bicistronic), which is the biological regulation basis to ensure efficient phosphorus removal.

Claims (7)

1. A strain of Citrobacter freundii transmuted with a phosphorus-accumulating gene, characterized in that it uses Citrobacter freundii as a host and introduces the host's own polyphosphate kinase Ppk1. 2. the Citrobacter freundii of transfer phosphorus accumulation gene according to claim 1, is characterized in that, only has a kind of polyphosphate kinase gene of Ppk1 in the genomic DNA of described Citrobacter freundii, and this Ppk1 The regulation mode of the gene and the exopolyphosphatase gene Ppx on the genome is bicistronic co-transcription. 3. the Citrobacter freundii of transfer phosphorus accumulation gene according to claim 1, is characterized in that, described Citrobacter freundii is Citrobacter freundii ATCC 8090, and described polyphosphate kinase Ppk1 The GenBank number of the gene is ANAV01000007.1. 4. the method for constructing the Citrobacter freundii of the transgenic phosphorus-accumulating gene of claim 1, is characterized in that, comprises the steps: (1) extracting the genomic DNA of Citrobacter freundii; (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification obtains the polyphosphate kinase Ppk1 gene of its own origin; (3) The polyphosphate kinase Ppk1 gene obtained in step (2) is connected to the T-Vector pMD19 carrier with T4 ligase to obtain the T-Ppk1 plasmid; (4) Digest the T-Ppk1 plasmid and the broad host expression plasmid pBBR1MCS-2 with restriction endonucleases to obtain the Ppk1 gene fragment and linearized pBBR1MCS-2; (5) Ligate the Ppk1 gene fragment obtained in step (4) with the linearized pBBR1MCS-2 with T4 ligase to obtain the pBBR1MCS-Ppk1 plasmid connected with the Ppk1 gene; (6) Using Citrobacter freundii as the recipient bacterium, the pBBR1MCS-Ppk1 plasmid obtained in step (5) was introduced into the recipient bacterium by electroporation to obtain Citrobacter freundii transgenic for the phosphorus accumulation gene. 5. the application of the Citrobacter freundii of transgenic phosphorus accumulation gene described in claim 1 in the phosphorus removal of waste water. 6. The application according to claim 5, wherein the phosphorus content in the wastewater is 0.1-20 mg/L. 7. A method for improving the ability of bacterial strains to accumulate phosphorus in wastewater, characterized in that the method is based on the bacterial strain with only Ppk1 polyphosphate kinase gene on the genome as the host bacterium, and the Ppk1 gene is in its genome in the host bacterium The regulatory mode of the upper and exopolyphosphatase gene Ppx is bicistronic co-transcription, the Ppk1 gene of the host bacteria is obtained by PCR, and constructed into an expression plasmid, and finally the expression plasmid connected with the Ppk1 gene is transformed into the Host bacterium, obtain the host bacterium of transgenic phosphorus accumulation gene, described host bacterium is: Acinetobacter baumannii, Clostridium acetobutylicum, Rhodospirillum, Nitrosomonas, Serratia or Shewanella bacteria.