Bacillus subtilis engineering bacteria with overexpression of pntab optimized by cofactor balance and application thereof

Abstract

This invention discloses a PntAB-overexpressing Bacillus subtilis engineered bacterium with cofactor balance optimization and its application, belonging to the field of agricultural technology. This Bacillus subtilis engineered bacterium contains... pntA Genes and pntB The gene was obtained by overexpressing a fragment derived from *Bacillus subtilis*. This invention involves overexpressing a gene derived from *Escherichia coli* in *Bacillus subtilis*. pntA Genes and pntB Genes were used to obtain recombinant strains that were able to regulate intracellular NADPH / NADP during fermentation. + The cofactor balance significantly improves the yield of 2,6-pyridinedicarboxylic acid (DPA), biofilm formation ability, and inhibitory activity against plant pathogenic fungi, providing efficient strain resources and synthetic biology construction strategies for the development of green biocontrol agents.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a PntAB-overexpressing Bacillus subtilis engineered strain with optimized cofactor balance and its application. Background Technology

[0002] Bacillus subtilis, as an important plant biocontrol bacterium, relies on its colonization ability on plant surfaces (mainly mediated by biofilms) and its ability to synthesize antibacterial metabolites for its application efficacy. 2,6-Pyridinedicarboxylic acid (DPA) is a natural product produced by Bacillus subtilis with broad-spectrum antifungal activity. Furthermore, the biofilm matrix of Bacillus subtilis provides it with physical protection and microecological advantages.

[0003] In existing technologies, homologous recombination technology has been used to replace the promoter of the DPA synthase gene, enabling its expression during the vegetative phase of the cell. Vegetative cells can then continuously secrete DPA, and existing literature reports that this technology can simultaneously increase DPA production and biofilm formation capacity. However, such methods may lead to uneven cellular metabolic burden or physiological imbalance. Cofactor levels are a crucial link connecting central metabolism, secondary metabolite synthesis, and cellular stress responses. In Bacillus subtilis, the reaction of DPA synthesis from glucose can be represented as: Glucose + HCO3- − + ATP + 2NADPH + 3NAD + + NH3→ DPA + ADP+ 2NADP + + 3NADH + Pi + 5H2O + 3H + It can be seen that the synthesis of DPA consumes ATP, NADPH, and NAD. + Furthermore, they cannot be regenerated. ATP / ADP, redox cofactors (NADP(H) and NAD(H)), and functional group carriers are cofactors involved in numerous intracellular metabolic reactions; changes in cofactor concentrations can have a wide-ranging impact on metabolism. Nicotinamide nucleotide transhydrogenase PntAB catalyzes the NADH + NADP reaction. + ⇌ NAD + The reversible NADPH reaction is a key enzyme regulating the intracellular NADPH / NADH balance. In metabolic engineering, PntAB has been used to address cofactor limitations and increase the yield of certain target chemicals, but its application in systematically reshaping the multifunctional phenotype of biocontrol bacteria, particularly in simultaneously enhancing biofilm and crop therapeutic functions, has not yet been reported.

[0004] Therefore, developing new strategies and strains that can synergistically optimize the multiple biocontrol functions of Bacillus subtilis through core metabolic engineering (cofactor balance regulation) is of great significance for developing the next generation of efficient and stable microbial agricultural inputs. Summary of the Invention

[0005] One objective of this invention is to provide an engineered Bacillus subtilis strain containing a nucleotide sequence as shown in SDQID NO.1. pntA The gene and nucleotide sequences are shown in SDQ ID NO.2. pntB The gene was obtained by transforming an overexpressed fragment of Bacillus subtilis.

[0006] The engineered bacteria are Escherichia coli (E. coli) Escherichia coli The two subunits of nicotinamide nucleotide transhydrogenase, PntA and PntB (hereinafter referred to as PntAB), were transferred into Bacillus subtilis (Bacillus subtilis). Bacillus subtilis )168 or its derivative strains were obtained, said pntA Genes and pntB The gene comes from E. coli ( Escherichia coli The GenBank serial numbers are 946628 and 946144, respectively.

[0007] Furthermore, the Bacillus subtilis strain is Bacillus subtilis strain 168 or uses a promoter. spoVG Alternative spoVFA The recombinant strain of Bacillus subtilis 168.

[0008] The second objective of this invention is to provide a method for constructing the above-mentioned engineered Bacillus subtilis strain, comprising the following steps: Step 1, pntA Genes and pntB Gene linking pntAB Gene coding sequence; Step 2, pntAB Genes were cloned into expression vectors to construct recombinant expression vectors; Step 3: Transform the recombinant expression vector into Bacillus subtilis host, screen for positive transformants, and obtain the genetically engineered bacteria.

[0009] Furthermore, the expression vector is plasmid pMA5.

[0010] The third objective of this invention is to provide the application of the above-mentioned engineered Bacillus subtilis strain in the prevention and control of plant pathogenic fungi.

[0011] The fourth objective of this invention is to provide the application of the above-mentioned engineered Bacillus subtilis strain, its fermentation broth or fermentation extract in the preparation of plant pathogenic fungi control products.

[0012] The engineered bacteria of this invention are Escherichia coli (E. coli) Escherichia coli The two subunits of nicotinamide nucleotide transhydrogenase, PntA and PntB (hereinafter referred to as PntAB), were transferred into Bacillus subtilis (Bacillus subtilis). Bacillus subtilis)168 or its derivative strains were obtained, said pntA The nucleotide sequence of the gene is shown in SEQ ID NO.1. pntB The nucleotide sequence of the gene is shown in SEQ ID NO.2. This invention utilizes the expression vector pMA5 to overexpress a gene derived from Escherichia coli MG1655 in Bacillus subtilis. pntAB Genes were used to obtain recombinant strains that were able to regulate intracellular NADPH / NADP during fermentation. + By balancing cofactors, the yield of 2,6-pyridinedicarboxylic acid (DPA), biofilm formation ability, and inhibitory activity against plant pathogenic fungi were significantly improved, providing efficient strain resources and synthetic biology construction strategies for the development of green biocontrol agents.

[0013] Compared with the prior art, the present invention has the following significant advantages: 1. Innovative Strategy: The strategy was first proposed and experimentally verified through overexpression... pntAB Gene regulation of the intracellular cofactor (NADPH / NADH) balance in Bacillus subtilis can serve as a core metabolic engineering strategy to systematically optimize multiple agronomic traits of the strain.

[0014] 2. Synergistic effect: The engineered strains obtained in this invention (such as MT-pntAB) not only further improve DPA production compared to the parent strain (MT), but also significantly enhance their biofilm formation ability and biocontrol and therapeutic effects on crops, achieving a "metabolic-driven" multi-effect synergistic improvement.

[0015] 3. Functional Expansion: Due to the enhanced biofilm colonization ability and stress resistance, this engineered strain has a stronger survival and colonization ability on plants, thus exhibiting a superior plant disease control effect compared to its parent strain, achieving a leap from "biocontrol" to "prevention and treatment" dual-function bacterial agent.

[0016] 4. Great application potential: This strain and its construction strategy provide direct strain resources and technical pathways for developing a new generation of high-performance, multifunctional Bacillus subtilis microbial preparations, and have broad application prospects in green agriculture. Attached Figure Description

[0017] Figure 1 This is a vector map of the PntAB overexpression plasmid pMA5-pntAB used in Example 1.

[0018] Figure 2 This is a comparison of the fermentation performance of different strains in Example 1. (A) Growth curve (OD) 600 (B) DPA production, (C) biofilm formation (crystal violet stained OD) 575The results showed that the MT-pntAB strain performed best in all three indicators.

[0019] Figure 3 This is a graph showing the plate confrontation inhibition effect of different Bacillus subtilis strains on Fusarium solani in Example 2.

[0020] Figure 4 The images show the preventive and therapeutic effects of different treatments on edamame pods in Example 3. (A) Effect diagram of the prevention group, (B) Effect diagram of the treatment group. Detailed Implementation

[0021] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Bacillus subtilis in the present invention and embodiments ( Bacillus sublitis The 168 (ATCC 23857) strain (labeled as WT strain) has been extensively disclosed in the prior art and is one of the most commonly used type strains of Bacillus subtilis.

[0025] Bacillus subtilis in the present invention and embodiments ( Bacillus sublitis MT strain is spoVF The construction method of the engineered strain with promoter replacement has been described in the literature "(1) Toya Y, Hirasawa T, Ishikawa S, Chumsakul O, Morimoto T, Liu S, Masuda K, Kageyama Y, Ozaki K, Ogasawara N, Shimizu H. Enhanced dipicolinic acid production during the stationary phasein Bacillus subtilisby blocking acetoin synthesis[J]. Bioscience Biotechnologyand Biochemistry, 2015, 79: 2073-2080. (2) Wang T, Wang XC, Han MH, Song XG,Yang DJ, Wang SY, Laborda P, Shi XC. Enhanced spoVF operon increases hostattachment and biocontrol ability of Bacillus subtilis for the managementof Ceratocystis fimbriata As reported in "in sweet potato[J]. Biological Control, 2021, 161:104651," the construction method is the traditional homologous recombination, which is simple to operate.

[0026] Bacillus subtilis in the present invention and embodiments ( Bacillus sublitis WT-PntAB is a compound found in Bacillus subtilis ( Bacillus sublitis The overexpression in strain 168 originated from Escherichia coli (E. coli). Escherichia coli MG1655 nicotinamide nucleotide transhydrogenase PntAB, which contains two subunits, PntA and PntB, is one of the most commonly used model strains of *Escherichia coli*. The *Bacillus subtilis* strain used in this invention and examples... Bacillus sublitis MT-PntAB is a compound found in Bacillus subtilis (…). Bacillus sublitis PntAB was overexpressed in the MT strain.

[0027] Fusarium tumefaciens in the present invention and its embodiments ( Fusarium ipomoeae NJC91 is a pathogen isolated from edamame pods in our laboratory. It has been described in the literature "Tang LQ, Chen X, Jiang YH, Cao JJ, Wu T, Wang XC, Shen Y, Zhang XT, Liu SY, Wang SY, Laborda P. Biocontrol of the zearalenone-producer Fusarium ipomoeae on soybean pods using anavirulent Lasiodiplodia theobromaeThis was reported in *isolate. Pest Manag Sci. 2025 Dec;81(12):8462-8476. doi: 10.1002 / ps.70150.”. It is available to the public from the Agricultural Microbiology Application Technology Team of Nantong University. Example 1

[0028] Construction and basic phenotypic validation of PntAB-overexpressing Bacillus subtilis engineered strain 1.1 Strains and Plasmids Host bacteria: Bacillus subtilis 168 wild-type (WT) and existing strains spoVF Promoter replacement strain (MT).

[0029] Expression vector: cloned from Escherichia coli pntA , pntB Genes (GenBank sequence numbers: 946628, 946144) were ligated using the overlap PCR method to form... pntAB The gene was amplified and purified using the high-fidelity DNA polymerase Phanta Max Super-Fidelity. The pMA5 plasmid was used as a vector, and double digestion at its BamHI and NdeI sites linearized the plasmid. During amplification... pntAB In the primer design for the gene, homologous arm sequences corresponding to both ends of the linearized plasmid were introduced. Finally, the gene fragment was ligated to the vector using homologous recombination to construct the target expression plasmid pMA5-pntAB. The map of the constructed overexpression plasmid is shown below. Figure 1 As shown.

[0030] pntA The nucleotide sequence is shown below:

[0031] pntB The nucleotide sequence is shown below:

[0032] 1.2 Construction of PntAB overexpression strains The pMA5-pntAB plasmid was introduced into WT and MT strains respectively by chemical transformation. After screening for kanamycin resistance and verification by colony PCR and sequencing, engineered strains WT-pntAB and MT-pntAB were obtained.

[0033] 1.3 Growth curves, DPA yield, and biofilm determination Four bacterial strains, WT, WT-pntAB, MT, and MT-pntAB, were inoculated into LB medium containing 20 g / L glucose and cultured at 37°C with shaking at 200 rpm.

[0034] Growth curve: OD was measured every 2 hours. 600 Growth curves were plotted from 0 to 24 h. The results showed that the growth rate and final biomass of strain MT-pntAB were significantly higher than those of the other three strains in the late logarithmic phase. Figure 2 (A)

[0035] DPA production: Samples were taken at 12 h, 16 h, and 20 h of fermentation, and the supernatant was collected by centrifugation. The DPA concentration was determined by HPLC. The results showed that the MT-pntAB strain had the highest DPA production, reaching 7.41 mM at 20 h, which was significantly higher than that of the MT strain (…). Figure 2 (B)

[0036] Biofilm formation: Four strains of bacteria were cultured in test tubes using liquid culture medium, and the bacterial culture was adjusted to OD. 600 =0.1, after static incubation for 72 h, staining was performed. The crystal violet was dissolved in 40% methanol and 10% glacial acetic acid aqueous solution and stained before OD was measured. 575 The results showed that the biofilm formation rate (OD) of the MT-pntAB strain was [data missing]. 575 The highest was approximately 2.2 times that of the WT strain. Figure 2 (C)

[0037] This embodiment demonstrates that overexpression of PntAB on the basis of MT strain (with enhanced DPA synthesis) can further synergistically promote cell growth, DPA synthesis and biofilm formation. Example 2

[0038] Determination of the in vitro inhibitory activity of engineered strains against plant pathogenic fungi 1.1 Test pathogens Fusarium oxysporum ( Fusarium ipomoeae NJC91 causes important diseases such as root rot in legumes.

[0039] 1.2 Flat Plate Standoff Experiment A 5 mm diameter Fusarium tumefaciens mycelium cake was inoculated in the center of a PDA plate, and four strains of Bacillus subtilis from Example 1 were streaked parallel to the cake at a distance of 3.5 cm. Plates without bacterial inoculation served as controls. The plates were incubated at 28°C for 5 days, and the colony radius of the pathogens was measured to calculate the inhibition rate.

[0040] The results show that ( Figure 3 All Bacillus subtilis treatments inhibited the growth of Fusarium solani, with the inhibition effect showing a gradient: MT-pntAB > MT > WT-pntAB > WT. Example 3

[0041] Evaluation of the in vivo disease control effect of engineered strains on edamame 1.1 Prevention and Treatment Trial Design Prevention experiment: Healthy edamame were surface-sterilized with 75% ethanol and then soaked in the following treatments for 1 hour.

[0042] 1) Sterile water (CK); 2) WT bacterial suspension (OD) 600 =1.0); 3) WT-pntAB bacterial suspension (OD 600 =1.0); 4) MT bacterial suspension (OD) 600 =1.0); 5) MT-pntAB bacterial suspension (OD) 600 =1.0).

[0043] Five pods were processed per group. Four hours after soaking, the outer skin was scratched, with the wounds kept to be 1 cm deep. 2 On the left and right sides, inoculate the wound with 20 μL of Fusarium solani spore suspension (1×10⁻⁶). 6 (spores / mL), and cultured in an artificial climate chamber for 5 days.

[0044] Treatment experiment: Healthy edamame beans were surface-sterilized with 75% ethanol, and the outer skin was scratched, with the wound controlled to be within 1 cm. 2 Around 10 μL of Fusarium spore suspension (1×10⁻⁶) was inoculated at the wound site. 6 (Spores / mL), after being cultured in an artificial climate chamber for 1 day, were taken out and 20 μL of each was used to treat the wound, and then placed back into the artificial climate chamber for 4 days.

[0045] 1.2 Observation Index The culture was completed on the 5th day after inoculation, and the following indicators were recorded: 1. Incidence rate (%): Count the number of pods with visible lesions in each group. Incidence rate = (Number of diseased pods / Total number of pods in the group) × 100%.

[0046] 2. Lesion diameter (cm): The diameter of the lesion at the wound site of each pod was measured using the cross-sectional method, and the average value was taken.

[0047] 3. Disease control effect: Calculate the inhibition rate of each strain treatment on the disease. Disease control effect = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100%.

[0048] 1.3 Results The results show that ( Figure 4 The effects of different engineered strains on the prevention and treatment of Fusarium diseases in edamame and sweet potato varied significantly.

[0049] 1. Preventive effect In the prevention experiment, the disease incidence rate in the sterile water control group (CK) was as high as 100%, with large areas of water-soaked rot appearing on the surface of the pods. Compared with the control group, each strain treatment group showed varying degrees of disease prevention effect. Among them, the MT-pntAB strain treatment group showed the best disease prevention effect, with a disease incidence rate of only 20%, while the WT strain and WT-pntAB strain treatment groups showed some prevention effect, but the effect was weaker than that of the MT modified strain. The results indicate that the MT-pntAB strain can more effectively colonize and inhibit subsequent pathogen infection during the prevention stage.

[0050] 2. Treatment efficacy In the treatment experiments, under the condition of inoculation with the pathogen followed by drug treatment, the disease progression in all treatments was higher than in the prevention experiments. The MT-pntAB strain treatment group still showed the best therapeutic effect, significantly inhibiting the spread of lesions. The MT strain treatment group also showed some therapeutic activity, while the therapeutic effects of the WT and WTP strains were relatively limited.

[0051] 3. Comprehensive Analysis Based on the combined results of preventive and therapeutic experiments, strain MT-pntAB exhibited the strongest and most stable antiviral activity against Fusarium oxysporum on live bean pods. Its protective effect in preventive treatments and its therapeutic efficacy in the later stages of infection were superior to the starting strains WT and MT, which is consistent with... pntAB The introduction of the gene may have enhanced the strain's adaptability to the plant microenvironment or led to the production of more antibacterial active substances.

Claims

1. An engineered Bacillus subtilis strain, characterized in that, Include nucleotide sequences as shown in SDQ ID NO.1 pntA The gene and nucleotide sequences are shown in SDQ ID NO.

2. pntB The gene was obtained by transforming an overexpressed fragment of Bacillus subtilis.

2. The engineered Bacillus subtilis strain according to claim 1, characterized in that, The Bacillus subtilis strain is Bacillus subtilis strain 168 or uses a promoter. spoVG Alternative spoVFA The recombinant strain of Bacillus subtilis 168.

3. The method for constructing the engineered Bacillus subtilis strain according to claim 1 or 2, characterized in that, Includes the following steps: Step 1, pntA Genes and pntB Gene linking pntAB Gene coding sequence; Step 2, pntAB Genes were cloned into expression vectors to construct recombinant expression vectors; Step 3: Transform the recombinant expression vector into Bacillus subtilis and screen for positive transformants, which are the engineered bacteria.

4. The construction method according to claim 3, characterized in that, The expression vector is plasmid pMA5.

5. The application of the engineered Bacillus subtilis strain according to claim 1 in the prevention and control of plant pathogenic fungi.

6. The use of the engineered Bacillus subtilis strain as described in claim 1, its fermentation broth or fermentation extract in the preparation of plant pathogenic fungi control products.

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