Artificial phosphorus-solubilizing module and application thereof in improving nitrogen-fixing and phosphorus-solubilizing capabilities of nitrogen-fixing microorganisms

Abstract

The invention relates to an artificial phosphate-solubilizing module and application thereof in improving the nitrogen-fixing and phosphate-solubilizing capabilities of nitrogen-fixing microorganisms. The artificial phosphate solubilizing module sequentially comprises an inducible promoter element and a conservative acid phosphatase gene acpA from a pseudomonas fluorescens strain from upstream to downstream. The artificial phosphate solubilizing module is inserted into a preset target site of a host microorganism chromosome in a homologous recombination manner, so that a host has the characteristic of efficiently expressing acid phosphatase, and the organic phosphorus degradation capacity and nitrogen fixation capacity of the host are remarkably improved. The method has high application value in the field of preparation of high-efficiency microbial fertilizer products. The method opens up a new path for agricultural green sustainable development, and has extremely important scientific significance and wide application prospects.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and more specifically, to an artificial phosphorus-solubilizing module and its application in enhancing the nitrogen fixation and phosphorus-solubilizing capabilities of nitrogen-fixing microorganisms. Background Technology

[0002] Nitrogen and phosphorus are the main nutrients affecting crop growth. Compared with traditional chemical nitrogen fertilizers, biological nitrogen fixation is not only lower in cost but also has less environmental impact, making it an environmentally friendly and efficient method of nitrogen supply with broad application prospects in agricultural production. Furthermore, the available phosphorus content in soil is low, and utilizing the metabolic functions of phosphorus-solubilizing microorganisms to drive the conversion of insoluble phosphorus into available phosphorus is an important way to improve soil phosphorus turnover and supply levels.

[0003] However, the phosphorus-solubilizing ability of rhizosphere nitrogen-fixing bacteria isolated so far is generally weak. Therefore, there is an urgent need for synthetic biology techniques to construct engineered bacteria that have both nitrogen-fixing and phosphorus-solubilizing functions. Summary of the Invention

[0004] The purpose of this disclosure is to provide an engineered bacterium that has both nitrogen fixation and phosphorus solubilization functions.

[0005] To achieve the above objectives, the first aspect of this disclosure provides an artificial phosphorus solubilization module, which comprises, from upstream to downstream, an inducible promoter element and a conserved acid phosphatase gene from *Pseudomonas fluorescens*. acpA .

[0006] Optionally, the nucleotide sequence of the artificial phosphorus solubilization module is shown in SEQ ID NO: 1.

[0007] Optionally, the nucleotide sequence of the inducible promoter element is the nucleotide sequence shown in positions 1-327 of SEQ ID NO: 1; The acid phosphatase gene acpA The nucleotide sequence is the nucleotide sequence shown in positions 328-2028 of SEQ ID NO: 1.

[0008] A second aspect of this disclosure provides a recombinant expression vector into which the artificial phosphorus solubilization module described in the first aspect is inserted.

[0009] The third aspect of this disclosure provides the application of the artificial phosphorus-solubilizing module described in the first aspect or the recombinant expression vector described in the second aspect in improving the nitrogen-fixing and phosphorus-solubilizing capabilities of nitrogen-fixing microorganisms.

[0010] Optionally, improving the phosphorus-solubilizing ability of nitrogen-fixing microorganisms includes improving their ability to degrade organic phosphorus; the nitrogen-fixing microorganism is *Pseudomonas schlegelii* A1501.

[0011] The fourth aspect of this disclosure provides a transformant in which the recombinant expression vector described in the second aspect is introduced; the host cell of the transformant is Pseudomonas schrenckii A1501.

[0012] The fifth aspect of this disclosure provides the application of the transformants described in the fourth aspect in promoting plant growth and in the preparation of microbial inoculants or microbial fertilizers.

[0013] Optionally, the plant includes rice and / or corn.

[0014] The sixth aspect of this disclosure provides a method for improving the nitrogen fixation and phosphorus solubilization capabilities of nitrogen-fixing microorganisms, the method comprising: The recombinant expression vector described in the second aspect is inserted at a specific site into the AmtB2 site of the chromosomal ammonium transporter of the nitrogen-fixing microorganism; wherein the nitrogen-fixing microorganism is Pseudomonas schrenckii A1501.

[0015] Through the above technical solution, this disclosure provides an artificial phosphorus-solubilizing module. By inserting the artificial phosphorus-solubilizing module into a predetermined target site on the host microbial chromosome through homologous recombination, the host acquires the characteristic of efficiently expressing acid phosphatase, thereby significantly improving the host's organophosphate degradation and nitrogen fixation capabilities. This has high application value in the preparation of high-efficiency microbial fertilizer products. It opens up a new path for the green and sustainable development of agriculture, possessing extremely important scientific significance and broad application prospects.

[0016] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 The recombinant expression vector pK18ms- described in this disclosure acpA A schematic diagram of the gene construction process, with arrows indicating the direction of gene transcription and insertion sites. Bam HI and Hin d Ⅲ.

[0018] Figure 2 The recombinant expression vector pK18ms- described in this disclosure acpA The PCR verification results.

[0019] Figure 3 The acid phosphatase gene described in this disclosure acpA PCR verification results of the successfully transformed nitrogen-fixing engineered bacteria A1591.

[0020] Figure 4 The host chassis bacterium described in this disclosure is *Pseudomonas aeruginosa*, a nitrogen-fixing bacterium. Pseudomonas stutzeri Analysis results of the organophosphorus hydrolysis capacity of A1501 and recombinant engineered bacteria A1591.

[0021] Figure 5 The host chassis bacterium described in this disclosure is *Pseudomonas aeruginosa*, a nitrogen-fixing bacterium. Pseudomonas stutzeri Analysis results of the biological nitrogen fixation capacity of A1501 and recombinant engineered bacteria A1591. Detailed Implementation

[0022] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0023] The "upstream and downstream" mentioned in this invention refer to the upstream and downstream directions of gene expression.

[0024] The first aspect of this disclosure provides an artificial phosphorus solubilization module, which, from upstream to downstream, comprises an inducible promoter element and a conserved acid phosphatase gene from *Pseudomonas fluorescens*. acpA .

[0025] In this disclosure, the inventors discovered a conserved acid phosphatase gene from *Pseudomonas fluorescens* strains. acpA The expression of acid phosphatase is regulated by inducible promoter elements. This disclosure describes the artificial synthesis of an inducible promoter and an acid phosphatase gene using chemical synthesis methods. acpA The constructed modular unit (i.e., the artificial phosphorus-solubilizing module) is inserted into a pre-defined target site on the host microbial chromosome via homologous recombination. This enables the host to efficiently express acid phosphatase, thereby significantly improving the host's organophosphate degradation and nitrogen fixation capabilities. It has high application value in the preparation of high-efficiency microbial fertilizer products.

[0026] In one embodiment of this disclosure, the nucleotide sequence of the artificial phosphorus solubilization module is shown in SEQ ID NO: 1. Specifically, the nucleotide sequence of the inducible promoter element is the nucleotide sequence shown in positions 1-327 of SEQ ID NO: 1; the acid phosphatase gene acpA The nucleotide sequence is the nucleotide sequence shown in positions 328-2028 of SEQ ID NO: 1.

[0027] A second aspect of this disclosure provides a recombinant expression vector into which the artificial phosphorus solubilization module described in the first aspect is inserted.

[0028] In this disclosure, artificial synthesis methods are used to add [materials] to both ends of the aforementioned artificial phosphorus solubilization module. Bam HI and Hind III restriction site, while utilizing Bam HI and Hind III. The PK18mob-sacB suicide plasmid was double-digested, and then the artificial combination module was ligated into the suicide plasmid pK18mob-sacB vector using a seamless cloning kit to obtain a homologous recombination expression vector.

[0029] The third aspect of this disclosure provides the application of the artificial phosphorus-solubilizing module described in the first aspect or the recombinant expression vector described in the second aspect in improving the nitrogen-fixing and phosphorus-solubilizing capabilities of nitrogen-fixing microorganisms.

[0030] In one embodiment of this disclosure, improving the phosphorus-solubilizing ability of nitrogen-fixing microorganisms includes improving their ability to degrade organic phosphorus; the nitrogen-fixing microorganism is *Pseudomonas schrenckii* A1501.

[0031] The fourth aspect of this disclosure provides a transformant in which the recombinant expression vector described in the second aspect is introduced; the host cell of the transformant is Pseudomonas schrenckii A1501.

[0032] In this disclosure, the acid phosphatase gene is expressed via the aforementioned recombinant expression vector. acpA The artificially combined module is transferred into the host microbial genome for expression. Specifically, the host is transformed using the above-mentioned recombinant expression vector via triparental conjugation to obtain the transformed organism, using wild-type nitrogen-fixing Pseudomonas stearothermia (…). Pseudomonas stutzeri A1501 is the host microorganism containing the recombinant plasmid pK18ms- acpA of E. coli Using DH5α strain as the donor and pRK2013 as the helper plasmid, a transformant expressing acid phosphatase with high efficiency was obtained. This transformant exhibits high nitrogen fixation and organophosphate degradation capabilities.

[0033] This disclosure provides a fifth aspect regarding the use of the transformants described in the fourth aspect in promoting plant growth and in the preparation of microbial inoculants or microbial fertilizers.

[0034] The transformed organism disclosed herein has a high capacity for hydrolyzing organophosphorus compounds, which can be used to prepare high-efficiency microbial agents and / or microbial fertilizers. These agents can alter soil properties, provide nitrogen and phosphorus nutrients to plants, and promote plant growth, thus having broad application prospects.

[0035] In one embodiment of this disclosure, the plant includes rice and / or corn.

[0036] The sixth aspect of this disclosure provides a method for improving the nitrogen fixation and phosphorus solubilization capabilities of nitrogen-fixing microorganisms, the method comprising: The recombinant expression vector described in the second aspect is inserted at a specific site into the AmtB2 site of the chromosomal ammonium transporter of the nitrogen-fixing microorganism; wherein the nitrogen-fixing microorganism is Pseudomonas schrenckii A1501.

[0037] In the above embodiments, inserting the recombinant expression vector disclosed herein into the preset target insertion site—the ammonium transporter AmtB2 site—can improve the nitrogen fixation capacity and phosphorus degradation capacity of nitrogen-fixing microorganisms, especially their ability to degrade organic phosphorus, without affecting the growth of the chassis strain.

[0038] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0039] Unless otherwise specified, all experimental conditions are based on conventional conditions known to those skilled in the art, such as those described in the "Sambrook" molecular cloning manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the manufacturer's recommendations.

[0040] Example 1 This example illustrates the acid phosphatase gene. acpA Recombinant expression vector pK18ms- acpA The construction of.

[0041] Recombinant expression vector pK18ms- acpA The construction diagram is as follows Figure 1 As shown.

[0042] First, the conserved acid phosphatase gene from *Pseudomonas fluorescens* was synthesized using artificial synthesis methods. acpA A phosphorus-solubilizing module, consisting of an inducible promoter element of 327 bp that regulates its expression, was then constructed. The artificial phosphorus-solubilizing module was then coupled to the ammonium transporter protein-coding genes at insertion sites via seamless cloning technology. amtB The upstream and downstream homologous arms of 2, and the nucleotide sequence of the artificial phosphorus solubilization module are shown in SEQ ID NO: 1.

[0043] Then use restriction endonucleases Bam HI and Hin d. The suicide plasmid pK18mob-sacB vector was double-digested. Using a seamless cloning kit (C115-02) purchased from Nanjing Novizan (Vazyme), the artificial conjugate module was integrated and ligated into the suicide plasmid pK18mob-sacB vector, and transformed into *E. coli* DH5α. Single colonies were obtained, and positive single colonies were selected for PCR verification. The results are as follows: Figure 2As shown, electrophoresis results indicated that positive colonies amplified a fragment approximately 3000 bp in size, demonstrating the successful construction of the recombinant expression vector pK18ms- acpA .

[0044] Example 2 This example illustrates the construction of the combined nitrogen-fixing recombinant engineered bacteria A1591.

[0045] The construction of recombinant strains utilizes the principle of homologous recombination. First, through triparental conjugation experiments, the recombinant expression vector pK18ms- was introduced into the strain. acpA Positive Escherichia coli DH5α donor bacteria, helper plasmid pRK2013, and recipient strain wild-type nitrogen-fixing Pseudomonas stearothermia ( Pseudomonas stutzeri A1501 was cultured overnight in LB liquid medium. The next day, 1 mL of the overnight culture was centrifuged at 6000 rpm for 5 min. The supernatant was discarded, and the bacterial pellet was resuspended in an equal volume of physiological saline and washed. The pellet was centrifuged again under the same conditions. The two-washed pellet was resuspended in 1 mL of physiological saline, mixed, and centrifuged again. 1 mL of the bacterial suspension was then spread onto antibiotic-free solid LB medium and incubated at 30°C for 2 days. After incubation, the plates were streaked onto plates containing 1 / 2 chloramphenicol and kanamycin. Once colonies appeared, single colonies were picked and streaked repeatedly. This purification process was repeated 2-3 times. The purified single colonies were then passed through a negative sieve containing 10% sucrose. sacB Strains that underwent homologous double crossover were obtained through lethal gene selection under sucrose selection pressure. Single colonies were selected for PCR verification, confirming the successful acquisition of recombinant engineered strain A1591. The results are as follows: Figure 3 As shown.

[0046] Example 3 This example illustrates the analysis results of the organophosphorus hydrolysis ability of the combined nitrogen-fixing recombinant engineered bacteria A1591.

[0047] The available phosphorus content of the supernatant of chassis strain A1501 and recombinant engineered strain A1591 in Monkina medium with lecithin as the sole phosphorus source was determined by molybdenum-antimony colorimetric assay to analyze the organic phosphorus hydrolysis capacity of the recombinant engineered strain. The specific method is as follows: The chassis strain A1501 and recombinant engineered strain A1591 were inoculated into Monkina medium with lecithin as the sole phosphorus source and cultured at 220 rpm and 30℃ for 4 days. Fermentation broth was collected every 24 hours, centrifuged at 8000 rpm for 10 min, and the supernatant was collected in a test tube. 2,4-Dinitrophenol was added and mixed well. Then, dilute sulfuric acid was added, and when the reaction solution became colorless and transparent, a colorimetric reagent was added. Finally, ultrapure water was added to bring the volume to a final level. The reaction was allowed to proceed for 30 min at room temperature, and the OD was measured. 700The value of is used to calculate the effective phosphorus content in the supernatant using the phosphorus standard curve.

[0048] The results are as follows Figure 4 As shown, the results indicated that there was no significant difference in available phosphorus content in the supernatant between A1591 and A1501 in the first 24 hours. From 48 to 96 hours, the available phosphorus content in the supernatant of A1591 was significantly higher than that of A1501, reaching a peak at 96 hours. The available phosphorus content in the supernatant of A1591 reached 2.87 mg / L, which is 1.178 times that of the wild-type strain A1501, indicating that the introduction of this module significantly improved the phosphorus-solubilizing ability of the chassis microorganisms.

[0049] Example 4 This example illustrates the results of the biological nitrogen fixation capacity analysis of the combined nitrogen-fixing recombinant engineered bacterium A1591.

[0050] The biological nitrogen fixation capacity of the recombinant engineered bacteria was analyzed by detecting the nitrogen fixation capacity of chassis strain A1501 and recombinant engineered bacteria A1591 using the acetylene reduction method. The specific method is as follows: Single colonies of freshly activated chassis strain A1501 and recombinant engineered strain A1591 were picked and inoculated into 20 mL of LB liquid medium, respectively, and cultured overnight at 220 rpm and 30°C. The next day, 10 mL of the overnight culture was centrifuged at 5000 rpm and 4°C for 10 min. After removing the supernatant, the cells were resuspended in 20 mL of nitrogen-free restriction K medium supplemented with lecithin and washed twice. The cells were then centrifuged for 10 min under the same conditions to obtain the washed cell pellet. The washed cell pellet was resuspended in LB liquid medium and the OD was adjusted. 600 Adjust the temperature to 1.0, and add 9 mL of nitrogen-free K-limiting medium and 1 mL of OD to a sterile 60 mL saline bottle. 600 The corresponding bacterial suspension with a value of 1.0 resulted in an initial OD... 600 The concentration was 0.1, and five replicates were performed for each sample. The sterile rubber stopper was tightened and the bottle was marked. The aluminum cap of each saline bottle was tightened with a capping tool to ensure the bottle was airtight. Argon gas was introduced into each saline bottle for 4 minutes to purge the air. Oxygen gas (0.5% of the bottle's internal space) and acetylene gas (10% of the bottle's internal space) were then introduced into each argon-filled bottle. The gas-filled saline bottles were placed in a shaker at 220 rpm and 30°C for incubation, and the bacterial enzyme activity was measured every 2 hours.

[0051] The results are as follows Figure 5As shown, the results indicated that the enzyme activity of the chassis strain A1501 was 4098 nmol ethylene / (mg protein·h), while that of the recombinant engineered strain A1591 was 5836 nmol ethylene / (mg protein·h). Significance analysis revealed that the nitrogenase activity of the recombinant engineered strain A1591 was significantly higher than that of the chassis strain A1501, demonstrating that the introduction of the artificial recombinant module in a nitrogen-free K-limited medium supplemented with lecithin can significantly improve the nitrogen-fixing capacity of the nitrogen-fixing microbial chassis.

[0052] Example 5 This example illustrates the analysis results of the rice growth-promoting ability of the combined nitrogen-fixing recombinant engineered bacteria A1591 under nitrogen-free conditions.

[0053] The plant fresh weight, plant dry weight, root fresh weight, and root dry weight of rice inoculated with strain A1501 and recombinant engineered strain A1591 were measured to analyze the rice growth-promoting ability of the recombinant engineered strain under nitrogen-free conditions. The control group (CK) was not inoculated with any strain. The specific method is as follows: The seeds of Zhonghua 11 rice were dehulled. Selected seeds were placed in a sterile 250mL Erlenmeyer flask, rinsed twice with sterile water, and then an appropriate amount of 75% alcohol was added. The flask was then shaken at 220rpm for 3 minutes. After discarding the alcohol, an appropriate amount of prepared 5% NaClO was added, and the flask was shaken at 220rpm for 10-12 minutes. This sterilization process was repeated once. The sterilized seeds were rinsed 6-8 times with sterile water, and then spread evenly on sterile filter paper and dried. The dried seeds were then evenly sown in half of MS semi-solid medium for germination. Germination was carried out at 30°C for 16 hours under light / 25°C for 8 hours in darkness, with a relative humidity of 80%, for 8 days.

[0054] In this study, diatomaceous earth (0.4 cm) and vermiculite were used as the substrate for rice growth. The two were mixed in a 1:1 volume ratio and sterilized at 121°C for 3 hours, then air-dried at room temperature. The mixture was then dispensed into flowerpots (8 cm in diameter and 10 cm in height), with each pot containing 250 g (dry weight). Eight days after rice germination, the MS medium attached to the rice seedlings was carefully washed off with sterile water. Four rice plants were then sown in each pot, and three pots were transplanted for each treatment. Two days after transplanting rice seedlings into flowerpots, as they adapt from the hydroponic environment to the soil-grown environment, the seedlings become sturdy and resilient. At this point, inoculation treatment can be performed. Single colonies of freshly activated tray strain A1501 and recombinant engineered strain A1591 are picked and inoculated into 100 mL of LB liquid medium, respectively, and cultured overnight at 220 rpm and 30°C. The next day, 10 mL of the overnight culture is centrifuged at 5000 rpm and 4°C for 10 min. After removing the supernatant, the cells are resuspended twice in 100 mL of nitrogen-free restriction K medium supplemented with lecithin and washed. The cells are then centrifuged for 10 min under the same conditions to obtain the washed cell precipitate. The washed cell precipitate is resuspended in LB liquid medium and the OD is adjusted. 600 Adjust to 1.0; absorb OD. 600 10 mL of a 1.0 μL bacterial suspension was inoculated around the roots of transplanted soil-grown rice seedlings. After inoculation, the rice plants were irrigated with a nitrogen-free hydroponic nutrient solution containing 320 mg / kg lecithin as the sole phosphorus source. In the fourth week after inoculation, the vermiculite and diatomaceous earth around the rice rhizosphere were cleaned off. Then, plant height, root length, plant fresh weight, root fresh weight, plant dry weight, and root dry weight were measured. Plant height and root length were measured using a meter stick method and recorded as plant height and root length. Plant fresh weight was measured by drying the roots and cutting them off. The above-ground and underground parts of the plant were weighed using an electronic balance and recorded as plant fresh weight and root fresh weight. Plant dry weight was measured by placing the plants in an oven at 105 °C for 15 minutes, then baking them at 70 °C until completely dry. The above-ground and underground parts of the plant were then weighed using an electronic balance and recorded as plant dry weight and root dry weight. The results are shown in Table 1.

[0055] Table 1

[0056] Compared with the control group, rice inoculated with chassis strain A1501 and recombinant engineered strain A1591 showed significant increases in plant fresh weight, plant dry weight, root fresh weight, and root dry weight. A1501 increased these values ​​by 13.48%, 19.88%, 34.69%, and 37.73%, respectively, while A1591 increased them by 18.32%, 25.10%, 54.74%, and 40.86%, respectively, demonstrating that the recombinant engineered strain A1591 has a stronger rice growth-promoting ability.

[0057] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0059] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. An artificial phosphorus catabolism module, characterized in that, The artificial phosphorus-solubilizing module comprises, from upstream to downstream, an inducible promoter element and an acid phosphatase gene from Pseudomonas fluorescens species conserved acpA .

2. The artificial phosphorus catabolism module of claim 1, wherein, The nucleotide sequence of the artificial phosphorus solubilizing module is shown in SEQ ID NO:

1.

3. The artificial phosphorus catabolism module of claim 2, wherein, The nucleotide sequence of the inducible promoter element is the nucleotide sequence shown in positions 1-327 in SEQ ID NO:

1. The nucleotide sequence of the acid phosphatase gene acpA is the nucleotide sequence shown as positions 328-2028 in SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that, The recombinant expression vector is inserted with the artificial phosphorus solubilizing module according to any one of claims 1-3.

5. The artificial phosphorus solubilizing module according to any one of claims 1-3 or the recombinant expression vector according to claim 4 is applied to improving the nitrogen fixation ability and phosphorus solubilizing ability of nitrogen-fixing microorganisms.

6. Use according to claim 5, wherein, Improving the phosphorus solubilizing ability of nitrogen-fixing microorganisms includes improving the ability of nitrogen-fixing microorganisms to degrade organic phosphorus. The nitrogen-fixing microorganism is Pseudomonas stutzeri A1501.

7. A transformant characterized in that, The transformant is introduced with the recombinant expression vector according to claim 4; and the host cell of the transformant is Pseudomonas stutzeri A1501.

8. The transformant according to claim 7 is applied to promoting plant growth and preparing microbial inoculants or microbial fertilizers.

9. Use according to claim 8, wherein, The plants include rice and / or corn.

10. A method for improving nitrogen fixation ability and phosphorus solubilization ability of a nitrogen-fixing microorganism, characterized by, The method comprises: The recombinant expression vector according to claim 4 is inserted into the chromosomal ammonium transporter AmtB2 site of the nitrogen-fixing microorganism; The nitrogen-fixing microorganism is Pseudomonas stutzeri A1501.

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