Application and method of methyltransferase in enhancing nitrogen-fixing capacity of nitrogen-fixing microorganisms

Abstract

The present disclosure relates to uses and methods of methyltransferase in enhancing the nitrogen-fixing ability of nitrogen-fixing microorganisms. The amino acid sequence of the methyltransferase is as shown in SEQ ID NO. 1. Further, the method comprises the steps of introducing a gene encoding a methyltransferase into a nitrogen-fixing microorganism and overexpressing it. According to the invention, the gene for coding the methyltransferase is introduced into the nitrogen-fixing microorganism and overexpressed, and the gene for coding the methyltransferase can specifically recognize the methylation site of the nitrogen-fixing gene of the nitrogen-fixing microorganism and perform specific modification, so that the expression quantity of the nitrogen-fixing gene of the nitrogen-fixing microorganism is improved, and the activity of the nitrogenase in the nitrogen-fixing microorganism is improved; the nitrogen fixation capability of nitrogen fixation microorganisms is further improved; particularly, the nitrogen fixation capability of the pseudomonas stutzeri A1501 is enhanced.

Description

Technical Field

[0001] This disclosure relates to the fields of microbiology and genetic engineering, and more specifically, to the use and methods of methyltransferases in enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms. Background Technology

[0002] To adapt to the complex and ever-changing external environment, microorganisms have developed sophisticated environmental adaptation mechanisms over a long period of evolution. One of the main genetic mechanisms is the regulation of gene expression through epigenetic modifications such as DNA methylation. The restriction modification (RM) system is one of the main forms of DNA methyltransferases in bacteria. The RM system mainly consists of DNA methyltransferases and restriction endonucleases (REases). Bacteria use the RM system to prevent the degradation of endogenous DNA and the invasion of exogenous DNA, thereby protecting the stability of their genetic makeup. In addition, the RM system also plays an important role in DNA mismatch repair and the regulation of gene expression.

[0003] Biological nitrogen fixation is an energy-intensive process, and the activity of nitrogenases is significantly affected by abiotic stress factors in the rhizosphere. Therefore, rhizosphere nitrogen-fixing microorganisms have developed a highly efficient and complex network for regulating nitrogen fixation gene expression over long-term evolution. However, whether DNA methyltransferases are involved in the regulation of nitrogen fixation gene expression is rarely reported. Therefore, identifying and utilizing DNA methyltransferases involved in the regulation of nitrogen fixation gene expression, and applying them to the modification of rhizosphere nitrogen-fixing microorganisms, is of great significance for the construction of efficient artificial nitrogen fixation systems. Summary of the Invention

[0004] The purpose of this disclosure is to provide the use and method of methyltransferases in enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms. The gene encoding the methyltransferase can specifically recognize the methylation site of the nitrogen fixation gene in nitrogen-fixing microorganisms, increase the expression level of the nitrogen fixation gene in nitrogen-fixing microorganisms, increase the activity of nitrogenase in nitrogen-fixing microorganisms, and thus improve the nitrogen fixation capacity of nitrogen-fixing microorganisms.

[0005] To achieve the above objectives, the first aspect of this disclosure provides the use of methyltransferases in enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Optionally, the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.2.

[0007] Optionally, the nitrogen-fixing microorganism is *Pseudomonas schlegelii* A1501.

[0008] Optionally, enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in the nitrogen-fixing microorganisms; preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

[0009] Optionally, the nitrogen fixation gene is nifH , nifD and nifK .

[0010] The second aspect of this disclosure provides a method for enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms, the method comprising the steps of: introducing a gene encoding a methyltransferase into nitrogen-fixing microorganisms and overexpressing it; The amino acid sequence of the methyltransferase is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.2.

[0011] Optionally, the gene encoding methyltransferase is introduced into nitrogen-fixing microorganisms via a recombinant expression vector; The recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown as positions 1-176 in SEQ ID NO. 3.

[0012] Optionally, the nitrogen-fixing microorganism is *Pseudomonas schlegelii* A1501.

[0013] Optionally, enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in the nitrogen-fixing microorganisms; preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

[0014] Optionally, the nitrogen fixation gene is nifH , nifD and nifK .

[0015] Through the above technical solution, this disclosure provides the use and method of methyltransferase in enhancing the nitrogen fixation ability of nitrogen-fixing microorganisms. This disclosure introduces the gene encoding methyltransferase into nitrogen-fixing microorganisms and overexpresses it. The gene encoding methyltransferase can specifically recognize the methylation site of the nitrogen fixation gene of nitrogen-fixing microorganisms and specifically modify it, thereby increasing the expression level of the nitrogen fixation gene of nitrogen-fixing microorganisms, increasing the activity of nitrogenase in nitrogen-fixing microorganisms, and thus improving the nitrogen fixation ability of nitrogen-fixing microorganisms; especially enhancing the nitrogen fixation ability of Pseudomonas schrenckii A1501.

[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 This is a diagram showing the homology analysis of genes encoding DNA methyltransferases.

[0018] Figure 2 It is a DNA methyltransferase encoding gene deletion mutant strain ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 Growth capacity analysis diagram.

[0019] Figure 3 It is a DNA methyltransferase encoding gene deletion mutant strain ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 (a bar chart of nitrogenase activity).

[0020] Figure 4 It is a DNA methyltransferase encoding gene deletion mutant strain ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 A bar chart showing the relative expression levels of nitrogen-fixing genes.

[0021] Sequence List Information SEQ ID NO.1: Amino acid sequence of methyltransferase.

[0022] SEQ ID NO.2: Nucleotide sequence of the gene encoding methyltransferase.

[0023] SEQ ID NO.3: Nucleotide sequence of a recombinant expression vector according to one embodiment of the present disclosure. Detailed Implementation

[0024] 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.

[0025] The first aspect of this disclosure provides the use of methyltransferases in enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms, the amino acid sequence of which is shown in SEQ ID NO.1.

[0026] The inventors of this disclosure, through extensive research, discovered that DNA methyltransferase domain analysis reveals a class I SAM-dependent methyltransferase domain. However, homology analysis with other proteins in this family shows a similarity of only 36%, suggesting it may be a novel DNA methyltransferase. The inventors of this disclosure, through third-generation single-molecule real-time sequencing, predicted that the gene encoding this methyltransferase can specifically recognize and modify the methylation site "ACAGNNNNNNRTCG" of nitrogen-fixing genes in nitrogen-fixing microorganisms, thereby increasing the expression level of nitrogen-fixing genes, enhancing the activity of nitrogenase in nitrogen-fixing microorganisms, and thus improving their nitrogen-fixing capacity. In particular, it can increase the expression level of nitrogen-fixing genes in *Pseudomonas stearothermiae* A1501, increase the activity of nitrogenase in *Pseudomonas stearothermiae* A1501, and thus enhance the nitrogen-fixing capacity of *Pseudomonas stearothermiae* A1501. In the methylation site "ACAGNNNNNNRTCG" of the nitrogen-fixing gene, N can be randomly selected from one of the nucleotide bases A, T, C, and G, and R can be randomly selected from nucleotide bases A or G.

[0027] According to this disclosure, the nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.2.

[0028] According to this disclosure, the nitrogen-fixing microorganism is *Pseudomonas schrenckii* A1501.

[0029] According to this disclosure, enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in nitrogen-fixing microorganisms; preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

[0030] According to this disclosure, the nitrogen-fixing gene is nifH , nifD and nifK .

[0031] The second aspect of this disclosure provides a method for enhancing the nitrogen fixation capacity of nitrogen-fixing microorganisms, the method comprising the steps of: introducing a gene encoding a methyltransferase into nitrogen-fixing microorganisms and overexpressing it; The amino acid sequence of the methyltransferase is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.2.

[0032] In this disclosure, the overexpression engineered strain A1501 constructed in this disclosure ( PST0633 The nitrogenase activity of the overexpressing engineered strain A1501 was increased by 40% compared to A1501. PST0633 The expression level of nitrogen-fixing genes in the sample was upregulated 7-8 times compared to that in A1501.

[0033] According to this disclosure, the gene encoding methyltransferase is introduced into nitrogen-fixing microorganisms via a recombinant expression vector; wherein, the gene encoding methyltransferase is introduced into nitrogen-fixing microorganisms using methods conventionally employed by those skilled in the art, such as a triparental conjugation method.

[0034] According to this disclosure, the recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown as positions 1-176 in SEQ ID NO.3.

[0035] According to this disclosure, the nitrogen-fixing microorganism is *Pseudomonas schrenckii* A1501.

[0036] According to this disclosure, enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in nitrogen-fixing microorganisms; preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

[0037] According to this disclosure, the nitrogen-fixing gene is nifH , nifD and nifK .

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

[0039] Unless otherwise specified, the raw materials, reagents, instruments and equipment involved in the embodiments of this disclosure can all be obtained by purchase.

[0040] Unless otherwise specified, the experimental conditions in this disclosure are conventional conditions well known to those skilled in the art, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer.

[0041] Example 1 This example illustrates the analysis of DNA methyltransferase sequence homology.

[0042] (1) Experimental method: The DNA methyltransferase gene was analyzed using the CDD domain database (https: / / www.ncbi.nlm.nih.gov / cdd), the sequence alignment analysis website ClustalW (https: / / www.genome.jp / tools-bin / clustalw), and ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / ESPript / index.php). PST0633Perform bioinformatics analysis.

[0043] (2) Experimental results and conclusions: Gene encoding DNA methyltransferase PST0633 It was identified as a class I SAM-dependent methyltransferase through domain prediction analysis, and sequence alignment with DNA methyltransferases of the same gene family showed a similarity of 36%. Figure 1 As shown, the gene encoding DNA methyltransferase PST0633 It was identified as a novel DNA methyltransferase in the Restriction Modification System Class I methyltransferase family.

[0044] Example 2 This embodiment is used to illustrate the construction. PST0633 Deletion mutant strain.

[0045] (1) Experimental method: The constructed DNA methyltransferase encoding gene PST0633 The main principle behind deletion mutants is homologous recombination of DNA.

[0046] The specific method is: PCR amplification to obtain the gene PST0633 Upstream and downstream homologous fragments (each over 500 bp) were ligated to an antibiotic resistance cassette (spectruminant antibiotic resistance cassette, purchased from Thermo Fisher Scientific) to obtain the recombinant fragment; the fragment was then ligated using restriction endonucleases. EcoR I and BamH I. The suicide vector pK18mobsacB was double-digested, and then the recombinant fragment was ligated into the suicide vector pK18mobsacB using a seamless cloning kit (Vazyme) to construct the recombinant vector. This vector was then transformed into *Pseudomonas stearothermiae* (A1501) using a triparental conjugation method. At this point, a single crossover occurs, integrating the vector into the whole genome. Single-crossover mutants are screened based on the vector's own resistance. During the second crossover, the suicide vector... sacB When cut from the genome, it carries a portion of the gene from a single crossover, resulting in two products: a deletion mutant and a wild-type strain, in a ratio of approximately 1:1. Double crossover mutants can be screened using 10% sucrose.

[0047] (2) Experimental results: Verified by PCR sequencing PST0633 The gene was successfully knocked out and named ΔPST0633 .

[0048] Example 3 This embodiment is used to illustrate the construction. PST0633 Deletion mutants, functional complements, and genes PST0633 Overexpression of recombinant engineered strains.

[0049] Experimental methods: Complete amplification was achieved through PCR. PST0633 Genes, using broad-host plasmids pLAFR3 ,choose BamH I and Hind The gene was double-digested at the III restriction site, and the gene was cloned using a seamless cloning kit (Vazyme). PST0633 Homologous recombination was performed with the enzyme-digested vector pLAFR3, and finally, single colonies were obtained by transforming E. coli. Single colonies were picked and PCR was performed to verify the successful acquisition of the DNA methyltransferase recombinant plasmid pLA. -PST0633 .

[0050] Donor bacteria E.coli (PLAFR3-PST0633), co-plasmid pRK2013, recipient bacteria: ΔPST0633 Alternatively, culture A1501 overnight in LB medium. Collect 1 mL of the overnight culture, centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend in an equal volume of physiological saline, centrifuge again, resuspend the bacterial pellet after two washes in 1 mL of physiological saline, centrifuge again, resuspend the bacterial cells in 50 µL of physiological saline, and drop onto antibiotic-free solid plates. Incubate the plates at 30°C. After 24 h, streak the plates on 1 / 2 chloramphenicol, spectinomycin, and spectinomycin-kanamycin plates, respectively. Pick single colonies for verification; if sequencing is correct, the bacteria have been successfully isolated. PST0633 Deletion mutants, functional complementation strains, and overexpression engineered strain A1501 ( PST0633 ).

[0051] Example 4 This example illustrates the overexpression engineered strain A1501 ( PST0633 Growth capacity analysis.

[0052] (1) Experimental method: Detection of deletion mutants in LB medium ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501, overexpression engineered strain A1501 ( PST0633 The growth status of the strain was analyzed to determine whether DNA methyltransferases affected its growth. The specific steps are as follows: (a) Deletion mutant strain ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 Incubate in LB liquid medium at 220 rpm and 30°C overnight; (b) Centrifuge the bacterial cells at 5000 rpm for 10 min the next day; (d) Resuspend the bacterial cells in physiological saline and wash the bacterial cells twice; (e) Suspend the bacterial cells in LB medium and adjust OD. 600 Up to 1.0; (f) Inoculate the bacterial cells into LB medium and culture them; (g) Measurement of deletion mutants using a fully automated growth curve instrument ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 Growth curve in LB medium, parameter settings, 30℃, OD measured every 4 hours. 600 .

[0053] (2) Experimental results and conclusions: like Figure 2 As shown, in LB medium, the engineered strain A1501 was overexpressed ( PST0633 The growth trend is similar to that of the chassis strain A1501. Therefore, in LB medium, the gene encoding DNA methyltransferase... PST0633 It does not affect the growth of the strain.

[0054] Example 5 This example illustrates the overexpression engineered strain A1501 ( PST0633 Nitrogenase activity assay.

[0055] (1) Experimental method: Deletion mutants were detected by acetylene reduction method. ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 The nitrogen-fixing ability of nitrogen is demonstrated through the following steps: (a) Select fresh, activated deletion mutant strains ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 Single colonies of the bacteria were inoculated into 20 mL of fresh LB liquid medium and incubated overnight at 220 rpm and 30°C.

[0056] (b) The next day, the bacterial culture was centrifuged at 5000 rpm and 4°C for 10 min. After removing the supernatant, the bacterial cells were resuspended in 20 mL of nitrogen-free limiting K medium and washed twice. The culture was then centrifuged for 10 min under the same conditions.

[0057] (c) OD of the washed bacterial solution 600 Adjust to 1.0, add 9 mL of nitrogen-free K-limiting medium and 1 mL of OD24-dimethylformamide to a sterile 60 mL saline bottle. 600 The bacterial culture was 1.0, with an initial OD of [value missing]. 600 Set the value to 0.1 and perform three parallel replicates for each sample.

[0058] (d) Tighten the sterile black rubber stopper, mark the bottle body, and use a capping tool to press the aluminum cap of each saline bottle to ensure the bottle is sealed.

[0059] (e) Pour argon into each saline bottle for 4 minutes to purge the air from the bottle, and then inject oxygen (0.5% of the bottle's space) and acetylene (10% of the bottle's space) into each bottle filled with argon.

[0060] (f) Place the gas-filled saline bottle in a shaker at 220 rpm and 30°C and measure the enzyme activity every 2 hours.

[0061] (2) Experimental results and conclusions: like Figure 3 As shown, compared with the chassis strain A1501, the overexpression of engineered strain A1501 ( PST0633 The nitrogenase activity of the enzyme increased by 40%. This indicates that the gene encoding DNA methyltransferase... PST0633 It can significantly improve the nitrogen fixation ability of chassis strain A1501.

[0062] Example 6 This example illustrates the overexpression engineered strain A1501 ( PST0633 Analysis of nitrogenase gene expression levels.

[0063] (1) Experimental methods Deletion mutant strains were analyzed using qRT-PCR. ΔPST0633, PST0633 Deletion mutant with functional complementation, chassis strain A1501 and overexpression engineered strain A1501 ( PST0633 Nitrogen fixation gene ( nifH , nifD , nifK The expression level of the compound under nitrogen-fixing conditions was analyzed.

[0064] (2) Experimental results and conclusions: like Figure 4 As shown, compared with the chassis nitrogen-fixing bacteria A1501, the overexpression of engineered strain A1501 ( PST0633 Nitrogen fixation gene nifH , nifD , nifK The expression levels increased by 7-fold, 7.5-fold, and 8.5-fold, respectively, under nitrogen-fixing conditions. This indicates that the expression levels of the DNA methyltransferase-encoding gene increased under nitrogen-fixing conditions. PST0633 It can increase the expression level of nitrogen-fixing genes after transcription.

[0065] 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.

[0066] 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.

[0067] 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. The use of methyltransferases in enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms, characterized in that, The amino acid sequence of the methyltransferase is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein, The nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.

2.

3. The use according to claim 1, wherein, The nitrogen-fixing microorganism is Pseudomonas schrenckii A1501.

4. The use according to any one of claims 1-3, wherein, Enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in them. Preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

5. The use according to claim 4, wherein, The nitrogen fixation gene is nifH , nifD and nifK .

6. A method for enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms, characterized in that, The method includes the following steps: introducing a gene encoding a methyltransferase into a nitrogen-fixing microorganism and overexpressing it; The amino acid sequence of the methyltransferase is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.

2.

7. The method according to claim 6, wherein, The gene encoding methyltransferase was introduced into nitrogen-fixing microorganisms via a recombinant expression vector; The recombinant expression vector contains a constitutive promoter; the nucleotide sequence of the constitutive promoter is shown as positions 1-176 in SEQ ID NO.

3.

8. The method according to claim 7, wherein, The nitrogen-fixing microorganism is Pseudomonas schrenckii A1501.

9. The method according to any one of claims 6-8, wherein, Enhancing the nitrogen-fixing capacity of nitrogen-fixing microorganisms includes increasing the activity of nitrogenase and the expression level of nitrogen-fixing genes in nitrogen-fixing microorganisms; Preferably, the methyltransferase specifically recognizes and modifies the methylation sites of nitrogen-fixing genes.

10. The method according to claim 9, wherein, The nitrogen fixation gene is nifH , nifD and nifK .

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