CN111286481B - Pseudomonas stutzeri mutant strain and construction method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a mutant strain, and a construction method and application thereof. The mutant strain of pseudomonas stutzeri is pseudomonas stutzeri DSM4166-TP (nifR), and is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 12 months and 20 days, and the preservation number is as follows: CGMCC No.19081. The invention has the beneficial effects that: compared with the existing wild DSM4166 strain, the mutant strain of Pseudomonas stutzeri has the advantages that the expression level of the azotase synthetic gene is greatly improved, and the activity of the produced azotase is greatly higher than that of the wild DSM4166 strain. The mutant strain of pseudomonas stutzeri of the invention can be produced in large scale in industry, and has wide commercial value.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a mutant strain, and a construction method and application thereof.
Background
Pseudomonas stutzeri DSM4166 (Pseudomonas stutzeri DSM4166, previously known as CMT.9. A) is a functional strain with nitrogen fixation ability, which was isolated from the rhizosphere of German Sorghum (Sorghum nutans) in 1897. This bacterium has been deposited at the Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH, germany Collection of microorganisms, with the number DSM4166. The bacteria can convert nitrogen in the air into ammonium which can be directly utilized by plants under ammonia-free and microaerobic conditions. At present, the work of genome sequencing of the strain is completed, and a section of Nitrogen-fixing gene island (Nif) is found to be contained on the genome of the strain, is positioned between cobs (PSTAA 1334) and gshp (PSTAA 1391) genes, has the length of 49kb, contains 58 different genes, and is a Nitrogen-fixing type with complete function and structure in the evolution of a biological Nitrogen-fixing system.
Because the nitrogen-fixing enzyme activity of the pseudomonas stutzeri DSM4166 is not high at present and does not reach satisfactory commercialization degree, the biological nitrogen-fixing way is further artificially designed and reconstructed and optimized by utilizing genetic engineering means, means such as simplification of nitrogen-fixing gene clusters, high-strength promoter replacement and the like, the nitrogen-fixing capacity of the biological nitrogen-fixing way is greatly improved, the natural nitrogen-fixing limit is strived to be broken through, and the satisfactory commercialization degree is reached.
Disclosure of Invention
The invention aims to provide a mutant strain of pseudomonas stutzeri, which has stronger biological nitrogen fixation capacity compared with the existing mutant strain of pseudomonas stutzeri. Meanwhile, the invention also provides a construction method and application of the mutant strain.
In order to achieve the purpose of the invention, the invention provides a mutant strain of Pseudomonas stutzeri, which is Pseudomonas stutzeri DSM4166-TP (nifR), is preserved in China general microbiological culture Collection center (CGMCC) at 04.12.2019, and has the preservation number: CGMCC No.19081.
The address of the depository: the institute of microbiology, national academy of sciences No. 3, xilu No.1, beijing, chaoyang, beijing.
The mutant strain DSM4166-TP (nifR) is an engineering strain constructed by inserting a gene rearranged and high-strength promoter into a reconstructed nitrogen fixing gene cluster nifR and inserting the gene cluster nifR into the genome of wild-type DSM4166 bacteria in a transposition mode. Based on the above, the present invention also provides a method for constructing the above mutant strain DSM4166-TP (nifR), comprising:
screening for a strong constitutive promoter in Pseudomonas stutzeri DSM 4166;
reconstructing a nitrogen fixation gene cluster nifR by using the screened strong constitutive promoter, and constructing a nitrogen fixation gene cluster plasmid p15A-cm-nif refactoring;
inserting a conjugative transfer element oriT and a transposition element TnpA into the nitrogen-fixing gene cluster plasmid p15A-cm-nif refecting;
the reconstructed nitrogen-fixing gene cluster is inserted into the genome of Pseudomonas stutzeri DSM4166 by means of conjugal transfer and transposition.
As a preferred mode of the present invention, the selection step of the strong constitutive promoter is:
measuring the growth curve of P.stutzeri DSM4166 under different culture conditions;
selecting 3 time points for transcriptome sequencing under each culture condition, and selecting from the transcriptome data genes whose expression level is 5% cut off in the first place;
excluding promoters with the length less than 100bp from the selected genes, and constructing a promoter detection plasmid of pBBR1-kan-4166 promoter-firefly;
the constructed plasmid was electrically transferred to p.stutzeri DSM4166, luciferase activity was detected, and a strong constitutive promoter was selected.
And (4) screening the promoter with higher promoter strength according to the luciferase activity detection result for reconstructing the nitrogen fixation gene cluster.
The invention also provides the application of the mutant strain DSM4166-TP (nifR) in preparing microbial fertilizers, such as nitrogen fixation fertilizers.
The invention has the beneficial effects that: compared with the existing wild DSM4166 strain, the mutant strain of Pseudomonas stutzeri has the advantages that the expression level of the azotase synthetic gene is greatly improved, and the activity of the produced azotase is greatly higher than that of the wild DSM4166 strain. The mutant strain of pseudomonas stutzeri of the invention can be produced in large scale in industry, and has wide commercial value.
Drawings
Figure 1 is a growth curve of p.stutzeri DSM4166 under different culture conditions;
FIG. 2 is a promoter screening plasmid map;
FIG. 3 shows luciferase activity assays;
figure 4 is a distribution of nitrogen fixation gene clusters in p.stutzeri DSM 4166;
FIG. 5 shows a reconstructed form of genes involved in biosynthesis of nitrogenase in the examples;
FIG. 6 shows the verification results of the first step of plasmid construction; from left to right, the five operon validation results are shown in FIG. 5;
FIG. 7 is a comparison map before and after the reconstruction of the nitrogen-fixing gene cluster;
FIG. 8 is ApaLI enzyme digestion verification map of azotobacter cluster plasmid p15A-cm-nif refactoring;
FIG. 9 is a diagram showing the sequencing result of the nitrogen-fixing gene cluster plasmid p15A-cm-nif refactoring;
FIG. 10 shows PCR verification of the adaptor colonies. PC p15A-oriT-TnpA-kan-nif refactoring, NC DSM4166 WT (wild type), 1-6 different zygotes;
FIG. 11 shows the PCR results of the expression levels of the nitrogenase-synthesizing genes after reconstitution;
FIG. 12 is a schematic representation of operon integrity PCR primer positions after reconstitution;
FIG. 13 is PCR detection of operon integrity after reconstitution; from left to right are the 1-5 operon PCR results, respectively, the sizes are: 3884bp,4062bp,3480bp,1867bp,4614bp;
FIG. 14 is a schematic representation of the characterization of the reconstituted mutant azotase activity assay.
Detailed description of the preferred embodiments
In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The embodiment provides a Pseudomonas stutzeri (Pseudomonas stutzeri DSM 4166) mutant strain DSM4166-TP (nifR), which is biologically preserved in 04.12.2019 at the common microorganism center of China general microbiological culture Collection center with the preservation number of CGMCC No.19081 and the preservation unit address: the institute of microbiology, national academy of sciences No. 3, xilu No.1, beijing, chaoyang, beijing.
The mutant strain DSM4166-TP (nifR) of the example has the following biological and cultural properties:
gram-negative bacilli, milk yellow transparent colonies with nearly circular shape and the like, irregular edges, folds on the surface and flat colonies, and fermentation liquor of the colonies is earthy yellow after being cultured in a 30-DEG C liquid LB culture medium for 24 hours.
The mutant strain DSM4166-TP (nifR) of the embodiment is an engineering strain constructed by inserting a nitrogen-fixing gene cluster nifR reconstructed by gene rearrangement and high-strength promoter insertion into the genome of wild-type DSM4166 bacteria in a transposition mode.
Example 2 construction and screening of the engineered strain DSM4166-TP (nifR) comprises the following steps:
1. screening for a Strong constitutive promoter in Pseudomonas stutzeri DSM4166
Culturing Pseudomonas stutzeri DSM4166 in LB medium at 30 deg.C and 37 deg.C under 4 different culture conditions; PMM medium, 30 ℃, 37 ℃ culture.
P. stutzeri DSM4166 growth curves were measured under 4 culture conditions, as shown in figure 1.
Selecting 3 time points for transcriptome sequencing under each culture condition, specifically:
TABLE 1 selection of time points for transcriptome sequencing under different culture conditions
The genes whose expression level was 5% first, cut off, were picked from the transcriptome data, as shown in Table 2.
TABLE 2P. stutzeri DSM4166 genes with higher transcription levels under different culture conditions
For the gene promoter with higher transcription level in the above Table 2, excluding the promoter with length less than 100bp, 25 strong constitutive promoters were selected in total to construct a promoter detection plasmid of pBBR1-kan-4166promoter-firefly, as shown in FIG. 2. And the promoter of the gene for resistance to genta is taken as a positive control, and the promoter is not taken as a negative control.
The constructed promoter detection plasmid is electrically transferred into P.stutzeri DSM4166, and the activity of luciferase is detected, wherein the specific detection steps are as follows:
(1) P.stutzeri DSM4166 containing promoter selection plasmid was inoculated in 1.8ml of LB liquid (containing Kanamycin) and cultured overnight at 30 ℃ and 950 rpm;
(2) Centrifugally collecting thalli from the overnight cultured bacterial liquid, washing twice with PBS buffer solution, and re-suspending the thalli with the PBS buffer solution;
(3) Measuring OD600 of the bacterial liquid after the heavy suspension, and diluting the OD600 to 1.0;
(4) Using a kit (
Single-Luciferase (Firefox) Reporter Assay Kit) is used for cracking the bacterial liquid, 50 mu l of cracking solution and 20 mu l of bacterial liquid are used, and the bacterial liquid is cracked for 20min by shaking at room temperature and 9 rpm (50 rpm);
(5) Luciferase activity assay was performed: mu.l of reaction substrate + 20. Mu.l of lysate were mixed well and the fluorescence value was measured by the instrument (since the reaction was faster, one set of reaction systems was added and then measured). The luciferase activity measured is shown in FIG. 3.
And screening the first 10 promoters with higher promoter strength according to the luciferase activity detection result for reconstructing the nitrogen-fixing gene cluster.
2. Reconstruction of nifR (nitrogen fixation gene cluster)
The distribution of the nitrogen-fixing gene cluster in wild type Pseudomonas stutzeri DSM4166 is shown in FIG. 4. In this example, genetic engineering techniques were used to reconstruct the gene cluster.
(1) The reconstruction strategy is: the nitrogen-fixing gene clusters are arranged according to the diagram shown in figure 5, and are respectively placed at the downstream of a screened strong constitutive promoter to form 5 operons, each operon is cloned to a p15A-cm vector, and 40bp homologous arms are arranged among the constructed operons for subsequent assembly.
(2) The reconstruction method comprises the following steps:
a. verifying the constructed operon: respectively extracting plasmids, enzyme digestion and verifying each operon
Enzyme digestion step: the constructed plasmids containing 5 operons respectively were extracted and digested with PvuII, pvuII, apaLI, ecoRV and PstI in the order shown in FIG. 5, the digestion system is shown in Table 3:
TABLE 3 enzyme digestion System
| Plasmid | 4ul |
| 10X cutsmart buffer | 1ul |
| Enzyme | 0.2ul |
| ddH 2 O | 4.8ul |
| Total | 10ul |
And (3) carrying out gel electrophoresis detection after enzyme digestion is carried out for 2h at 37 ℃, wherein the detection results are shown in FIG. 6, and five operon verification result graphs of FIG. 5 are respectively shown from left to right, and it can be seen from the graphs that 5 operons are correctly enzyme-digested.
b. And (3) cutting gel by enzyme to recover each operon, and assembling 5 operons into a complete nitrogen fixation gene cluster through combined action of Gibson assembly and GB05-dir in vivo assembly. As shown in FIG. 7, 5 strong promoters were inserted into the assembled nitrogen-fixing gene cluster compared to that before reconstitution.
c. Enzyme digestion verification of the completely reconstructed azotobacter cluster plasmid p15A-cm-nif refactoring:
(1) Extracting a plasmid p15A-cm-nif refactoring, and carrying out enzyme digestion verification by using ApaLI (ApaLI), wherein an enzyme digestion system is shown in a table 4:
TABLE 4 enzyme digestion System
| Plasmid | 4ul |
| 10X cutsmart buffer | 1ul |
| ApaLI | 0.2ul |
| ddH 2 O | 4.8ul |
| Total | 10ul |
(2) The enzyme was cleaved at 37 ℃ for 2h and then detected by gel electrophoresis, the results of which are shown in FIG. 8. DNA sequencing was performed by sequencing Inc. and the reconstructed nif gene cluster was free of point mutations as shown in FIG. 9.
d. The conjugative transfer element oriT and the transposable element TnpA are inserted into the reconstituted plasmid.
3. Inserting the reconstructed nitrogen-fixing gene cluster into the genome of P.stutzeri DSM4166 by means of conjugal transfer and transposition to obtain the engineering bacterium DSM4166-TP (nifR).
4. Colony PCR to verify whether the reconstructed nitrogen-fixing gene cluster is inserted into P.stutzeri DSM4166 genome, the colony PCR result of the engineering bacteria DSM4166-TP (nifR) is shown in FIG. 10, PC: p15A-oriT-TnpA-kan-nif refectoring, NC: DSM4166 WT (wild type), 1-6: a different linker.
5. Determination of expression level of reconstructed nitrogenase-synthesizing gene
(1) The cell culture was performed according to the procedure for measuring the nitrogenase activity.
(2) Respectively at the point of 99% Ar +1% 2 Samples were taken at 4H and 16H later, and samples were taken at 4H (20H) after 16H injection of C2H2 to extract total RNA, with triplicate experiments per group. Reverse transcription and real-time fluorescent quantitative analysis are carried out, and three times of repeated experiments are carried out by qPCR.
A qPCR primer was designed inside nifH (ferritin synthesis gene) gene with GAPDH as internal reference gene.
As shown in FIG. 11, by the detection of real-time fluorescent quantitative PCR, compared with the wild-type DSM4166 bacteria, the transcription level of nifH gene of the engineering bacteria DSM4166-TP (nifR) is significantly and greatly improved, along with the increase of the culture time of the bacteria, the transcription level of nifH gene is linearly increased, and when the bacteria are cultured for 20h, the transcription level of nifH gene is improved by 60 times.
6. Integrity detection of post-reconstruction operators
Taking the cDNA after reverse transcription as a template, as shown in FIG. 12, taking an upstream primer in the first CDS of each operon and a downstream primer in the last CDS, and detecting whether the operon is complete by PCR, thereby excluding the possibility that the operon contains a terminator. As shown in FIG. 13, the PCR results of the 1-5 operons from left to right are respectively: 3884bp,4062bp,3480bp,1867bp,4614bp.
7. Determination of nitrogenase Activity after reconstitution
(1) Inoculating the engineering bacterium DSM4166-TP (nifR) into 1.8ml LB containing Kanamycin, culturing overnight at 30 ℃ and 950 rpm;
(2) Centrifuging at 11000rpm for 1min to collect thallus, washing with nitrogen-free medium 460 once, re-suspending thallus with 1ml of nitrogen-free medium 460, measuring OD600, and diluting to OD600=1.0;
(3) Adding 18ml of nitrogen-free medium 460 (K2 HPO4 0.5g/L, mgSO4x7H2O 0.2g/L, naCl 0.1g/L, yeast extract 0.2g/L, feCl3x6H2O 15mg/L, na2MoO4x2H2O 12mg/L, DL-Na-malate 6.6g/L pH 7.0) into an anaerobic triangular flask (100 ml), adding 2ml of bacteria solution of OD600=1.0 into the anaerobic flask, and mixing uniformly;
(4) Introducing mixed gas (99% argon and 1% oxygen) into the bottle for 3min, then covering the bottle cap tightly, and culturing at 30 ℃ and 200rpm for 16h;
(5) Then taking out 10ml of gas from the anaerobic bottle and injecting 10ml of acetylene gas inwards, and continuously culturing for 4 hours at 30 ℃ and 200 rpm;
(6) Performing gas chromatography detection (KB-Al 2O3/Na2SO4 capillary column with length of 30.0m, inner diameter of 0.53mm, film thickness of 20.0 μm, gasification chamber temperature of 100 deg.C, chromatography procedure of 100 deg.C, and FID detector for 5 min);
(7) And (3) measuring the protein content of the bacterial liquid after GC detection, and characterizing the azotobacter activity through ethylene yield/protein amount/time. The protein concentration determination method comprises the following steps: 1) Centrifugally collecting thalli in an anaerobic bottle, washing by ddH2O, and discarding supernatant; 2) Adding 500 μ l 0.5M NaOH solution, resuspending, boiling for 5min, and lysing thallus; 3) Adding 500 μ l of 0.5M HCl, mixing, and centrifuging at 13900rpm for 10min; 4) The supernatant was diluted appropriately and subjected to protein concentration determination (Coomassie brilliant blue standard curve method).
The nitrogenase activity was characterized as shown in figure 14. Compared with wild type DSM4166 azotase, the engineering bacteria DSM4166-TP (nifR) constructed by the invention has 14 times higher activity of azotase, the result is consistent with the transcription level result of nifH gene in the previous real-time fluorescent quantitative PCR detection, and the experimental result shows that the engineering bacteria DSM4166-TP (nifR) with higher azotase activity is obtained by a biological engineering means, thereby providing a good foundation for the future industrial development.
Claims (2)
1. A pseudomonas stutzeri strain characterized by: the strain is Pseudomonas stutzeri, is preserved in China general microbiological culture Collection center in 12.04.2019, and has the preservation number as follows: CGMCC No.19081.
2. Use of a pseudomonas stutzeri strain according to claim 1 for the preparation of a microbial fertilizer.
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