Engineering nitrogen-fixing vibrio natriivibrio as well as preparation method and application thereof

Abstract

The invention discloses an engineered nitrogen-fixing vibrio natrieae and a preparation method and application thereof, and relates to the field of synthetic biology, a nitrogen-fixing gene nif gene cluster is cloned and assembled on an escherichia coli-yeast shuttle vector to form recombinant plasmids, the plasmids are transformed and extracted by escherichia coli and then transformed into wild vibrio natrieae, and the engineered nitrogen-fixing vibrio natrieae is obtained. And obtaining the nitrogen-fixing vibrio natriivibrio. The invention further discloses the obtained engineered nitrogen-fixing vibrio natriticus and application thereof. The invention develops novel vibrio natriticus capable of efficiently fixing nitrogen in a broad spectrum as well as a preparation method and application of the novel vibrio natriticus.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to an engineered nitrogen-fixing nanofibrillator, its preparation method, and its applications. Background Technology

[0002] Nitrogen makes up 78% of the atmosphere and is essential for plant growth, being a key factor in achieving high and stable crop yields. However, most organisms cannot directly utilize atmospheric nitrogen; instead, they absorb it in the form of ammonia or ammonium salts. Increasing the availability of these forms of nitrogen can significantly improve crop yields, especially in poor soils. Currently, chemical nitrogen fertilizers are the main source of nitrogen for crops, but their low utilization efficiency and improper application can lead to eutrophication of water bodies, damage aquatic ecosystems, and ultimately threaten human health.

[0003] Biofertilizers, organic products containing microorganisms, offer a promising alternative to chemical nitrogen fertilizers, partially or completely replacing them. Biofertilizers are cost-effective and versatile, increasing crop yields while reducing fertilizer and energy consumption. However, the nitrogen-fixing microorganisms in biofertilizers face limitations in field application, including low nitrogen fixation efficiency, sensitivity to environmental conditions, and host specificity. These challenges hinder their competitiveness and stability, limiting the widespread adoption of biological nitrogen fixation in agriculture. Improving the efficiency of biofertilizers and enhancing their nitrogen-fixing capacity remain key objectives in this field.

[0004] To address these challenges, synthetic biology has emerged as a powerful approach, opening new possibilities for advancing biological nitrogen fixation. Advances in gene editing have provided precise tools for creating strains with enhanced nitrogen-fixing capabilities. *Vibrio natans* (…) Vibrio natriegens *Vibrio nabinoids* possesses advantages such as rapid growth, high salt tolerance, and the ability to utilize multiple carbon sources. During the logarithmic growth phase, the ribosome density of *Vibrio nabinoids* reaches 115,000 per cell, giving it enormous potential for heterologous protein expression. Furthermore, *Vibrio nabinoids* has a mature gene-editing system, making it an ideal chassis cell for nitrogen fixation. In summary, the construction of engineered nitrogen-fixing *Vibrio nabinoids* using synthetic biology is crucial for supporting the widespread application of biological nitrogen fixation in agriculture. It also has significant implications for increasing crop yields, reducing agricultural dependence on chemical fertilizers, and ensuring global food security and environmental sustainability.

[0005] Therefore, those skilled in the art are dedicated to developing a nanofibrillated bacterium capable of efficient spectral nitrogen fixation, its preparation method, and its applications. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a novel nanofibrillary bacterium capable of efficient and broad-spectrum nitrogen fixation, as well as its preparation method and application.

[0007] To achieve the above objectives, the present invention provides an engineered nitrogen-fixing nanofibrillator and its preparation method, characterized by comprising the following steps: Step 1: Cloning the nitrogen fixation gene; Step 2: The nitrogen-fixing genome is loaded into the E. coli-yeast shuttle vector to form a recombinant plasmid; Step 3: Transform the recombinant plasmid into Escherichia coli; Step 4: Extract plasmids from E. coli in Step 3 and extract nitrogen fixation gene fragments; Step 5: Transform the plasmid extracted in Step 4 into wild-type Nystatin to obtain nitrogen-fixing Nystatin.

[0008] In a preferred embodiment of the present invention, the nitrogen-fixing gene is nif A gene cluster, which clones the nitrogen-fixing gene by DNA synthesis or by amplifying a fragment from genomic DNA, excludes regions that do not affect nitrogenase activity and adds essential genes to the electron transport chain.

[0009] In another preferred embodiment of the present invention, the nitrogen-fixing gene comes from A. vinelandii DJ or P. stutzeri A1501 nif The gene clusters, with sequences shown in SEQ ID NO.1 or SEQ ID NO.2, respectively.

[0010] In another preferred embodiment of the present invention, step 2 specifically includes: connecting 2000 bp upstream and downstream homologous arms to the 5' and 3' ends of the nitrogen fixation gene fragment, the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and assembling the amplified fragment of the nitrogen fixation gene with the linearized vector pRS415 into a recombinant plasmid using a yeast assembly program.

[0011] In another preferred embodiment of the present invention, step 3 specifically includes: adding a recombinant plasmid containing a vector fragment and a DNA fragment to be assembled into yeast competent cells through chemical transformation of yeast, extracting the recombinant plasmid from yeast using the Zymoprep yeast mini-preparation kit and transforming it into Escherichia coli.

[0012] In another preferred embodiment of the present invention, step 4 specifically includes: isolating and purifying plasmids from Escherichia coli and performing sequencing and multiple enzyme digestion to verify correct assembly and sequence; storing Escherichia coli containing non-mutant plasmids; extracting plasmids; and isolating nitrogen-fixing gene fragments with natural Vibrio homologous arms by enzyme digestion.

[0013] In another preferred embodiment of the present invention, step 5 specifically includes: Wild-type Vibrio natans was streaked on LB3 solid medium and then transferred to a 30 ℃ incubator for about 8 h until a clear single colony was observed. Add LB3 medium and IPTG solution to a test tube, pick a single colony of wild-type Vibrio adenophora, and incubate it in a shaker at 30 ℃ for 4-4.5 h at 220 rpm. After the bacterial culture becomes turbid, measure its OD600. When the OD600 reaches 3.9 to 4.6, the competent cells can be used. A premixed system was prepared by adding nitrogen-fixing gene fragments, culture medium, IPTG solution, and competent cells, and then incubated at 30 ℃ for 6 h. Add 1 mL of LB3 medium for resuscitation, incubate at 30 ℃ and 220 rpm for 1.5 h, then spread the bacterial culture onto a plate and incubate at 30 or 37 ℃.

[0014] The present invention also provides engineered nitrogen-fixing nanofibrillator bacteria prepared by the above method.

[0015] 10. In a preferred embodiment of the present invention, the engineered solid-demand Vibrio nanobractea bacteria are respectively Vibrio natriegens W34 and Vibrio natriegens W78, with accession numbers CCTCC NO: M20252394 and CCTCC NO: M20252395, respectively, contains nitrogen-fixing gene sequences as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0016] The present invention also provides applications of the above-described engineered nitrogen-fixing nanofibrillator.

[0017] Technical effect

[0018] 1. This invention utilizes gene clusters derived from different hosts that possess highly efficient nitrogen-fixing capabilities ( nif Gene clusters), precisely integrated and optimized for expression in chassis microorganisms with superior physiological characteristics—Vibrio natans ( Vibrio natriegensIn this study, engineered, highly efficient, and broad-spectrum nitrogen-fixing strains were constructed. These engineered nitrogen-fixing *Vibrio natans* strains can serve as promising alternatives to chemical nitrogen fertilizers and are crucial for supporting the widespread application of biological nitrogen fixation in agriculture. They overcome efficiency bottlenecks and leverage the advantages of multi-source gene clusters. The extremely high ribosome density and rapid growth characteristics of *Vibrio natans* provide a powerful biosynthetic factory for nitrogenase, a complex protein complex with multiple subunits, high molecular weight, and high energy consumption, theoretically enabling protein expression levels and nitrogenase activity far exceeding those of natural nitrogen-fixing bacteria. Nitrogen fixation efficiency is significantly improved: the engineered strains exhibit nitrogenase activity and nitrogen fixation rates far exceeding those of natural nitrogen-fixing microorganisms, more effectively converting atmospheric nitrogen into plant-available ammonia nitrogen, better meeting the high-yield requirements of crops (especially non-leguminous crops). This provides a promising alternative that can partially or completely replace chemical nitrogen fertilizers. The method is cost-effective and versatile, while reducing fertilizer and energy consumption. The engineered nitrogen-fixing *Vibrio natans* strains developed in this invention achieve breakthrough performance improvements through synthetic biology strategies. Its core advantages lie in: utilizing the Vibrio natans ultra-high protein expression platform (ribosome density 115,000 / cell) to integrate multi-source nitrogen-fixing gene clusters, making nitrogenase activity 3-5 times that of traditional strains; combining the natural salt tolerance and rapid proliferation ability (doubling time <10 minutes) of the chassis bacteria, giving the engineered bacteria strong adaptability and soil colonization advantages in marginal lands such as saline-alkali land; combined with the carbon source utilization technology of industrial and agricultural waste, it can reduce production costs by more than 30% and reduce chemical nitrogen fertilizer by 30-50% per hectare, alleviating the environmental problems caused by the unreasonable application of chemical nitrogen fertilizer from the source.

[0019] 2. The present invention selects a suitable chassis that requires Vibrio natans (Nanobi). Vibrio natriegens This bacterium, with its rapid growth, high salt tolerance, ability to utilize multiple carbon sources, strong protein expression capacity, and mature gene editing system, is an ideal substrate cell for nitrogen fixation. Instead of introducing a single gene, a complete and functionally optimized nitrogen-fixing gene cluster from different nitrogen-fixing microorganisms is integrated into the *Vibrio natans* genome. These gene clusters contain the complete set of genes encoding the nitrogenase complex and its regulation, assembly, and cofactor synthesis. This enhances environmental adaptability. *Vibrio natans* naturally possesses high salt tolerance, enabling it to grow well in saline-alkali soils or soils with high irrigation water salinity, overcoming the survival barriers of most traditional nitrogen-fixing bacteria in such environments.

[0020] 3. The strain of this invention has the advantage of rapid growth: Its ultra-fast reproduction rate allows it to quickly form a dominant population at the colonization site, effectively resisting competitive pressure from indigenous microorganisms and improving survival rate and colonization density in the rhizosphere or soil. It can utilize a variety of inexpensive carbon sources (such as glycerol, glucose, sucrose, etc.), reducing application costs. It also exhibits strong environmental adaptability: Thanks to the high salt tolerance of the Vibrio natans chassis and the rapid growth characteristics of the engineered bacteria, this strain maintains good survival and nitrogen fixation capabilities in saline-alkali lands and arid / semi-arid regions, thus broadening its application range.

[0021] 4. The strains in this invention exhibit stable and reliable performance in the field: The engineered bacteria are highly competitive, have high colonization density, and are highly tolerant to the environment, significantly improving their survival rate, persistence, and stability of nitrogen fixation function in complex farmland environments.

[0022] 5. The method of this invention can effectively reduce reliance on chemical fertilizers and environmental pollution: The highly efficient and stable biological nitrogen fixation capacity can significantly replace the application of chemical nitrogen fertilizers, reduce agricultural production costs, and promote sustainable agricultural development.

[0023] 6. The methods and applications of this invention have a mature industrial foundation: *Vibrio natans* can be amplified at low cost using conventional fermentation equipment, utilizing inexpensive carbon sources (such as industrial glycerol and molasses) to achieve high-density fermentation in conventional fermenters (without the need for high-oxygen transfer equipment), requiring no special culture conditions; the engineered nitrogen-fixing strain has been verified by multiple passages of PCR, and the exogenously introduced nitrogen-fixing gene cluster is stably present on the chromosome of *Vibrio natans*; *Vibrio natans* is a GRAS (Generally Recognized as Safe) microorganism, with no reported pathogenicity, ensuring environmental safety and meeting the regulatory requirements for biological products. It can be combined with compound fertilizers to form high-value-added coupled fertilizers.

[0024] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0025] Figure 1 This is a nitrogenase activity test result of engineered nitrogen-fixing bacteria according to a preferred embodiment of the present invention; Figure 2 This is a test result of the growth capacity of engineered nitrogen-fixing bacteria according to a preferred embodiment of the present invention.

[0026] The strains and their preservation information in this invention are as follows: strains Vibrio natriegensW34, accession number CCTCC NO: M20252394, accession date: October 30, 2025, depositary institution: China Center for Type Culture Collection (CCTCC), address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, taxonomic name: sodium-dependent Vibrio, taxonomic name (Latin name): Vibrio natriegens; strains Vibrio natriegens W78, accession number CCTCC NO: M20252395, accession date October 30, 2025, depositary institution: China Center for Type Culture Collection (CCTCC), address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, taxonomic name: sodium-dependent Vibrio, taxonomic name (Latin name): Vibrio natriegens Detailed Implementation

[0027] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0028] This document provides a method for constructing engineered nitrogen-fixing nanofibrillary bacteria using synthetic biology and validates their functionality.

[0029] Example 1 Construction of engineered nitrogen-fixing nanofibrillary bacteria

[0030] Through selective design, natural clones from different species were created. nif Gene clusters, encompassing different bacterial orders, including Enterobacterales , Bacillales , Pseudomonadales and Rhizobiales To compare their relative performance in sodium-dependent Vibrio species. Natural nif Gene clusters are complex in composition. During the design process, regions that do not affect nitrogenase activity (such as the Pst1307-Pst1312 region in *P. stutzeri* A1501) are excluded, and essential genes in the electron transport chain (such as those in *A. vinelandii* DJ) are added. rnf / fix ).These nif Gene clusters are constructed from DNA synthesized or amplified fragments from genomic DNA, fused using yeast assembly, and cloned into an appropriate host plasmid backbone. The specific steps are as follows: 1. Collected from NCBI P. polymyxa WLY78 K. oxytoca M5al、 A. vinelandii DJ and P. stutzeriThe DNA sequence information of A1501 was obtained, and a 5kb fragment was synthesized de novo using gene synthesis to serve as a template for PCR amplification and assembly.

[0031] 2. Connect 150bp upstream and downstream homologous arms at the 5' and 3' ends of the gene cluster, and use a yeast assembly program to assemble the amplified fragment from the DNA fragment with the linearized vector into the E. coli-yeast shuttle vector pRS415.

[0032] 3. The assembled large-fragment plasmid was extracted from yeast and transformed into E. coli using the Zymoprep yeast small-scale preparation kit (Zymo Research, catalog number D2004).

[0033] 4. The plasmid was isolated and purified from E. coli and sequenced and digested with multiple enzymes to verify correct assembly and sequence. E. coli containing the non-mutant plasmid was stored for further experiments.

[0034] 5. Obtain the complete nitrogen-fixing gene cluster from the non-mutant plasmid in E. coli. The plasmid was obtained using the macherey-nagelnucleoBond BAC 100 kit (740579), and the nitrogen-fixing gene fragment with the homologous arm of the native Vibrio was isolated by enzymatic digestion.

[0035] 6. Wild-type Vibrio natans was streaked on LB3 solid medium and then transferred to a 30 °C incubator for about 8 hours until distinct single colonies were observed.

[0036] 7. In a 14 mL Falcon test tube, precisely add 2.997 mL of LB3 medium and 3 µL of 1 MIPTG solution, and pick a single colony of wild-type Vibrio nautiloides for culture. Incubate at 30 °C on a shaker for 4–4.5 h at 220 rpm.

[0037] 8. Measure the OD after the bacterial solution becomes cloudy. 600 , to OD 600 When the chromatogram (Ct) is 3.9–4.6, competent cells can be used. Take a 1.5 mL centrifuge tube (sterile) and add the nitrogen fixation gene fragment. Prepare a premixed system according to the ratio of 350 µL IOM medium + 0.35 µL 1 M IPTG + 3.5 µL competent cells per experimental group of the fragment to be transformed. Then, take 350 µL of the premixed system and mix it with the nitrogen fixation gene fragment. Incubate at 30 ℃ for 6 h.

[0038] Add 1 mL of LB3 medium for resuscitation, incubate at 30 ℃ and 220 rpm for 1.5 h, and then aspirate 20 µL to 100 µL of bacterial culture onto a plate (if the transformation efficiency is extremely low, increase to 1 mL; centrifuge and collect the supernatant to 100 µL before spreading). Incubate at 30 or 37 ℃. (IOM (Instant Ocean Medium) is a commercially available solid seawater culture medium for ornamental fish farming (Aquarium Systems Inc., catalog number SS15-10). Dissolve it in water at a concentration of 28 g / L and sterilize at 121 ℃ for 20 minutes before use.)

[0039] 9. From P. polymyxa WLY78 K. oxytoca M5al、 A. vinelandii DJ and P. stutzeri The nitrogen-fixing gene cluster of A1501 was inserted into engineered strains of Vibrio natans and named W1 / W2 / W34 and W78, respectively.

[0040] Example 2: Nitrogenase activity test of engineered nitrogen-fixing Vibrio natans

[0041] 1. Inoculate single colonies into sterile tubes with culture medium supplemented with appropriate antibiotics and incubate overnight in a 30°C incubator.

[0042] 2. After washing the bacterial solution twice with M9 inorganic salt medium, the overnight cultured strain was diluted with M9 medium and incubated overnight at 30°C in an incubator.

[0043] 3. After washing the bacterial solution twice with nitrogen-free M9 medium, dilute the culture to OD in nitrogen-inhibiting medium. 600 =0.4, and supplemented with appropriate antibiotics and 1.43 mM serine to promote nitrogenase inhibition.

[0044] 4. In glass vials with PTFE-silicone diaphragm screw caps, use a vacuum manifold to replace the top space of the vials with 100% argon gas. Inject acetylene into each culture flask at a concentration of 10% (v / v) to initiate the reaction.

[0045] 5. Acetylene reduction was carried out at 30°C for 20 hours, with shaking at 220 rpm in a shaking incubator to prevent cell aggregation, and then quenched by adding 0.5 ml of 4M NaOH to each vial.

[0046] 6. To quantify ethylene production, the headspace of the culture was removed using a gas-tight syringe through a rubber stopper and manually injected into a SHIMADZU GC2030 gas chromatograph. The ethylene production of the strain was quantified by the integrated peak area. See Appendix for details. Figure 1

[0047] Example 3: Growth capacity test of engineered nitrogen-fixing Vibrio nanofibrils

[0048] The engineered strain was incubated overnight in LB3 medium and washed three times with nitrogen-free M9 medium.

[0049] The strain was cultured in nitrogen-free M9 medium with an initial OD600 of 0.01. The strain was then placed in an anaerobic incubator, and its OD600 was measured periodically. See appendix for details. Figure 2

[0050] The present invention also includes engineered nitrogen-fixing Vibrio natans prepared by the above method and its applications, preferably including the application of the above strains in agriculture.

[0051] The applications of engineered nitrogen-fixing sodium-dependent Vibrio bacteria in agriculture mainly include: replacing or reducing the application of chemical nitrogen fertilizers (such as for rice, wheat, vegetables, and fruit trees), promoting crop growth and increasing yield and quality, improving soil fertility, and for organic agriculture and saline-alkali land improvement. The implementation methods typically involve: applying the inoculant to seed coatings, root dipping seedlings, mixing it with organic fertilizer for basal application, applying it as a top dressing through drip irrigation / fertigation, or foliar spraying to provide nitrogen nutrition for biological nitrogen fixation during the core crop growth stages.

[0052] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for constructing engineered nitrogen-fixing nanofibrillator bacteria, characterized in that, Includes the following steps: Step 1: Cloning the nitrogen fixation gene; Step 2: The nitrogen-fixing genome is loaded into the E. coli-yeast shuttle vector to form a recombinant plasmid; Step 3: Transform the recombinant plasmid into Escherichia coli; Step 4: Extract plasmids from E. coli in Step 3 and extract nitrogen fixation gene fragments; Step 5: Transform the plasmid extracted in Step 4 into wild-type Nystatin to obtain nitrogen-fixing Nystatin.

2. The method as described in claim 1, characterized in that, The nitrogen fixation gene is nif A gene cluster, which clones the nitrogen-fixing gene by DNA synthesis or by amplifying a fragment from genomic DNA, excludes regions that do not affect nitrogenase activity and adds essential genes to the electron transport chain.

3. The method as described in claim 1, characterized in that, The nitrogen fixation gene comes from A. vinelandii DJ or P. stutzeri A1501 nif The gene clusters, with sequences shown in SEQ ID NO.1 or SEQ ID NO.2, respectively.

4. The method as described in claim 1, characterized in that, Step 2 specifically includes: linking 2000 bp upstream and downstream homologous arms to the 5' and 3' ends of the nitrogen fixation gene fragment, the upstream and downstream homologous arm sequences being shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and assembling the amplified fragment of the nitrogen fixation gene with the linearized vector pRS415 into a recombinant plasmid using a yeast assembly program.

5. The method as described in claim 1, characterized in that, Step 3 specifically includes: adding a recombinant plasmid containing a vector fragment and a DNA fragment to be assembled into yeast competent cells through chemical transformation of yeast; extracting the recombinant plasmid from yeast using the Zymoprep yeast mini-production kit and transforming it into E. coli.

6. The method as described in claim 1, characterized in that, Step 4 specifically includes: isolating and purifying plasmids from Escherichia coli and performing sequencing and multiple enzyme digestion to verify correct assembly and sequence; storing Escherichia coli containing non-mutant plasmids; extracting plasmids; and isolating nitrogen-fixing gene fragments with homologous arms of natural Vibrio by enzyme digestion.

7. The method as described in claim 1, characterized in that, Step 5 specifically includes: Wild-type Vibrio natans was streaked on LB3 solid medium and then transferred to a 30 ℃ incubator for about 8 h until a clear single colony was observed. Add LB3 medium and IPTG solution to a test tube, pick a single colony of wild-type Vibrio natans for culture, and incubate in a shaker at 30℃ for 4-4.5 h at 220 rpm. After the bacterial culture becomes turbid, measure its OD600. When the OD600 reaches 3.9 to 4.6, the competent cells can be used. A premixed system was prepared by adding nitrogen-fixing gene fragments, culture medium, IPTG solution, and competent cells, and then incubated at 30 ℃ for 6 h. Add 1 mL of LB3 medium for resuscitation, incubate at 30 ℃ and 220 rpm for 1.5 h, then spread the bacterial culture onto a plate and incubate at 30 or 37 ℃.

8. Engineered nitrogen-fixing nanofibrillator prepared by the method according to any one of claims 1-8.

9. The engineered nitrogen-fixing nanofibrillator prepared by the method of claim 8, characterized in that, The engineered solid-demanding nanoviolet bacteria are respectively Vibrio natriegens W34 and Vibrio natriegens W78, with accession numbers CCTCC NO: M20252394 and CCTCC NO: M20252395, contains nitrogen-fixing gene sequences as shown in SEQ ID NO.1 or SEQ ID NO.2, respectively.

10. The application of the engineered nitrogen-fixing nanofibrillator as described in any one of claims 8-9.

Images