Method for improving the content of azotobacter vinelandii cell protein and application thereof

Abstract

The application provides a method for increasing the content of Azotobacter chroococcum cell protein and application thereof. The method inhibits or blocks the synthesis pathway of poly-beta-hydroxybutyric acid in the cell of the Azotobacter chroococcum, so that the metabolic flow and carbon source of the cell are more used for synthesizing nitrogen-containing biomass such as protein, the content of the cell protein is significantly increased, the metabolic cost of nitrogen fixation is reduced, and more protein is output under the same carbon source consumption.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and more specifically, to a method for increasing the protein content of brown azotocin bacteria and its application. Background Technology

[0002] Global food security is facing the dual pressures of population growth and resource constraints. The global population is projected to reach approximately 9.7 billion by 2050, leading to a surge in demand for high-quality protein. Traditional protein production models, reliant on resource-intensive livestock and aquaculture, are hampered by bottlenecks such as arable land per capita, dwindling freshwater resources, and overexploitation of wild fisheries. This not only exacerbates the "food-feed" competition but also threatens the stability of ecosystems and the global protein supply chain. Developing efficient, low-environmental-footprint alternative protein sources has become an urgent global issue.

[0003] Single-cell protein (SCP), a promising alternative, is obtained through the cultivation of microorganisms such as yeast, fungi, algae, or bacteria. It boasts core advantages including high resource conversion efficiency and independence from arable land and seasonal climate. It can be cultivated at high density using inexpensive substrates such as agricultural waste, yielding protein per unit area far exceeding that of traditional agriculture, demonstrating its commercial potential as a high-quality feed protein and even a human nutritional food. Among these, brown azotocin (… Azotobacter vinelandii Because of its aerobic nitrogen-fixing properties, it can directly use atmospheric nitrogen to synthesize amino acids and proteins without relying on industrial nitrogen fertilizers, which can reduce production costs and carbon emissions from the source, making it one of the preferred strains for SCP production.

[0004] In the field of single-cell protein metabolic engineering, rationally designing and deleting competing metabolic pathways to reprogram cellular carbon and energy flows to reduce resource dissipation of non-target products is a core strategy for increasing microbial protein content. This strategy has been validated in microorganisms such as *Fusarium verticillata*, significantly improving carbon conversion and protein synthesis efficiency. However, current technologies for directly producing SCPs using *Azotobacter spp.* still face the bottleneck of low bacterial protein content, limiting their economical and efficient application. Targeted technological optimization is urgently needed to unlock their potential as a sustainable protein source. Summary of the Invention

[0005] The purpose of this invention is to significantly increase the protein content of bacteria by deleting genes related to PHB synthesis and genes related to fluorescent siderophore synthesis through metabolic engineering strategies, thereby reconstructing the carbon, nitrogen, and energy metabolic flux of bacteria.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for increasing the protein content of brown azotocin bacteria, the method comprising inactivating the PHB synthesis pathway in the brown azotocin bacteria.

[0007] Optionally, the brown azotocin bacteria are those with preservation numbers GDMCC No. 1.1487, GDMCC No. 1.1412, GDMCC No. 1.1413, and GDMCC No. 1.1486.

[0008] Optionally, the PHB synthesis pathway inactivating the brown azotocin bacteria includes at least one of gene editing, gene silencing, and enzyme activity inhibition.

[0009] Optionally, the gene editing includes knocking out, mutating, or replacing key genes in the PHB synthesis pathway; The key genes include phbA , phbB , phbC At least one of the genes.

[0010] Preferably, the method includes removing the brown azotocin from the bacteria. phbA, phbB or phbC The gene was replaced with a resistance gene; Preferably, the resistance gene is Kan Gene.

[0011] On the other hand, the present invention provides the application of the above method in the production of single-cell proteins.

[0012] On the other hand, the present invention provides the application of the above method in the preparation of feed protein.

[0013] On the other hand, the present invention provides the application of the above method in the preparation of food raw materials.

[0014] Through the above technical solution, the present invention provides a method for increasing the protein content of brown azotocin bacteria and its application. This method, by inhibiting or blocking metabolic pathways that compete with bacterial protein synthesis in brown azotocin bacteria, directs more cellular metabolic resources, such as carbon, nitrogen and energy metabolism, toward protein synthesis, effectively increasing the bacterial protein content while reducing the metabolic cost of nitrogen fixation.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 It is wild-type GDMCC No. 1.1487 and Δ phbB Electrophoresis image of mutant strain colony PCR verification; Figure 2For wild-type GDMCC No. 1.1487 and Δ phbB viable cell count of mutant strain after 60 hours of culture; Figure 3 For wild-type GDMCC No. 1.1487 and Δ phbB Analysis of PHB content in mutant strains; Figure 4 For wild-type GDMCC No. 1.1487 and Δ phbB Comparison of nitrogenase activity in mutant strains; Figure 5 For wild-type GDMCC No. 1.1487 and Δ phbB Protein content of mutant strains; Figure 6 For wild-type GDMCC No. 1.1487 and Δ phbB The total protein content that a mutant strain can produce by consuming 1 g of carbon source; Figure 7 For wild-type GDMCC No. 1.1487 and Δ phbA Protein content of mutant strains; Figure 8 For wild-type GDMCC No. 1.1487 and Δ phbC Protein content of mutant strains; Figure 9 For wild-type GDMCC No. 1.1412 and Δ phbB Protein content of mutant strains; Figure 10 For wild-type GDMCC No. 1.1413 and Δ phbB Protein content of mutant strains; Figure 11 For wild-type GDMCC No. 1.1486 and Δ phbB Protein content of mutant strains; Figure 12 For wild-type GDMCC No. 1.1487 and Δ pvdD Electrophoresis image of mutant strain colony PCR verification; Figure 13 For wild-type GDMCC No. 1.1487 and Δ pvdD Comparison of the appearance of culture supernatant of mutant strains; Figure 14 For wild-type GDMCC No. 1.1487 and Δ pvdD UV-Vis absorption spectrum of culture supernatant of mutant strain; Figure 15 For wild-type GDMCC No. 1.1487 and Δ pvdD Growth curve of the mutant strain; Figure 16 For wild-type GDMCC No. 1.1487 and Δ pvdD Dry weight of mutant cells after 60 h of culture; Figure 17 For wild-type GDMCC No. 1.1487 and Δ pvdD Comparison of nitrogenase activity in mutant strains; [[ID=4l]]Figure 18 For wild-type GDMCC No. 1.1487 and Δ pvdD Protein content of mutant strains. Detailed Implementation

[0017] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0018] On one hand, the present invention provides a method for increasing the protein content of brown azotocin bacteria, the method comprising inactivating the PHB synthesis pathway in the brown azotocin bacteria.

[0019] Optionally, the brown azotocin bacteria are those with preservation numbers GDMCC No. 1.1487, GDMCC No. 1.1412, GDMCC No. 1.1413, and GDMCC No. 1.1486.

[0020] Optionally, the PHB synthesis pathway inactivation in the brown azotocin bacterium includes at least one of gene editing, gene silencing, and enzyme activity inhibition.

[0021] Optionally, the gene editing includes knocking out, mutating, or replacing key genes in the PHB synthesis pathway; The key genes include phbA , phbB , phbC At least one of the genes.

[0022] Optionally, the method includes removing the brown azotocin from the bacteria. phbA, phbB or phbC The gene was replaced with a resistance gene; Preferably, the resistance gene is Kan Gene.

[0023] On the other hand, the present invention provides the application of the above method in the production of single-cell proteins.

[0024] On the other hand, the present invention provides the application of the above method in the preparation of feed protein.

[0025] On the other hand, the present invention provides the application of the above method in the preparation of food raw materials.

[0026] The present invention will be further described in detail below through examples. All raw materials used in the examples are commercially available.

[0027] Example 1 This embodiment illustrates the construction and verification of a mutant strain of brown azotobacter with gene deletion.

[0028] (1) Strains and culture medium: Tested strains: Azotobacter brownigra GDMCC No. 1.1487, Azotobacter brownigra GDMCC No. 1.1412, Azotobacter brownigra GDMCC No. 1.1413 and Azotobacter brownigra GDMCC No. 1.1486; all of the above strains are deposited at the Guangdong Provincial Microbial Culture Collection Center.

[0029] Burk medium (Bueno Batista et al., 2021), with specific components and culture conditions shown in Table 1.

[0030] Table 1 Burk medium formulation

[0031] (2) Gene deletion mutations and primer design: Using *Azotocinus brownii* GDMCC No. 1.1487 as material, the kanamycin resistance gene was employed. Kan Replace it phbB Genes, construction phbB The following primers were designed and synthesized for gene deletion mutants: amplification phbB 800 bp homologous arm upstream of the gene: upstream primer phbB upF, downstream primer phbB upR (and) Kan Gene matching); amplification phbB 800 bp downstream homologous arm of gene: upstream primer phbB downF (and) Kan Gene matching), downstream primers phbB downR; amplification Kan Resistance gene: upstream primer phbB upKF (and) phbB upF matching), downstream primers phbB downKR (and) [[ID=--64]]phbB (downR matches).

[0032] Using the brown azotocin bacterium GDMCC No. 1.1487 as material, the kanamycin resistance gene was utilized. Kan Replace itphbA Genes, construction phbA Gene deletion mutant strains. The following primers were designed for amplification. phbA 800 bp homologous arm upstream of the gene: upstream primer phbA upF, downstream primer phbA upR (and) Kan Gene matching); amplification phbA 800 bp downstream homologous arm of gene: upstream primer phbA downF (and) Kan Gene matching), downstream primers phbA downR; amplification Kan Resistance gene: upstream primer phbA upKF (and) phbA upF matching), downstream primers phbA downKR (and) phbA (downR matches).

[0033] Using the brown azotocin bacterium GDMCC No. 1.1487 as material, the kanamycin resistance gene was utilized. Kan Replace it phbC Genes, construction phbC Gene deletion mutant strains. Design the following primers for amplification: phbC 800 bp homologous arm upstream of the gene: upstream primer phbC upF, downstream primer phbC upR (and) Kan Gene matching); amplification phbC 800 bp downstream homologous arm of gene: upstream primer phbC downF (and) Kan Gene matching), downstream primers phbC downR; amplification Kan Resistance gene: upstream primer phbC upKF (and) phbC upF matching), downstream primers phbC downKR (and) phbC (downR matches).

[0034] Using *Azotocinus brownii* GDMCC No. 1.1412 as material, the kanamycin resistance gene was utilized. Kan Replace it phbB Genes, construction phbB Gene deletion mutant strains. Design the following primers for amplification: phbB 800 bp homologous arm upstream of the gene: upstream primer phbB upF1, downstream primer phbB upR1 (matched with the Kan gene); amplification phbB800 bp downstream homologous arm of gene: upstream primer phbB downF1 (and) Kan Gene matching), downstream primers phbB downR1; amplification Kan Resistance gene: upstream primer phbB upKF1 (and) phbB upF1 matching), downstream primers phbB downKR1 (matches phbBdownR1).

[0035] Using *Azotocinus brownii* GDMCC No. 1.1413 as material, the kanamycin resistance gene was utilized. Kan Replace it phbB Genes, construction phbB Gene deletion mutant strains. Design the following primers for amplification: phbB 800 bp homologous arm upstream of the gene: upstream primer phbB upF2, downstream primer phbB upR2 (and) Kan Gene matching); amplification phbB 800 bp downstream homologous arm of gene: upstream primer phbB downF2 (matched to the Kan gene), downstream primer phbB downR2; amplification Kan Resistance gene: upstream primer phbB upKF2 (and) [[ID=---123]]phbB upF2 matching), downstream primers phbB downKR2 (and) phbB (downR2 match).

[0036] Using *Azotocinus brownii* GDMCC No. 1.1486 as material, the kanamycin resistance gene was utilized. Kan Replace it phbB Genes, construction phbB Gene deletion mutant strains. Design the following primers for amplification: phbB 800 bp homologous arm upstream of the gene: upstream primer phbB upF3, downstream primer phbB upR3 (and) Kan Gene matching); amplification phbB 800 bp downstream homologous arm of gene: upstream primer phbB downF3 (and) Kan Gene matching), downstream primers phbB downR3; amplification Kan Resistance gene: upstream primer phbB upKF3 (and) phbB upF3 matching), downstream primersphbB downKR3 (and) phbB (downR3 match).

[0037] Sequence 5' -> 3': phbBupF:ATGCTGAAGAGCACTTTTGAGATC,SEQ ID NO: 8;phbBupR:TTTCCCGTTGAATATGGCTCATAGATTTCCCTTCCTTTTTTGTC,SEQ ID NO: 9;phbBdownF:GATGCTCGATGAGTTTTTCTAACGGCATTCGGAAAGCTGCTAGG,SEQ ID NO: 10;phbBdownR:ATTACCGGCGGTGACGCTTCC,SEQ IDNO: 11;phbBupKF:GACAAAAAAGGAAGGGAAATCTATGAGCCATATTCAACGGGAAA,SEQ ID NO: 12;phbBdownKR:CCTAGCAGCTTTCCGAATGCCGTTAGAAAAACTCATCGAGCATC,SEQ ID NO:13;phbAupF:AATCAACGAATTGCATTCGTCA,SEQ ID NO:14;phbAupR:TTTCCCGTTGAATATGGCTCATGAAGGAATTACCTCATATGAAT,SEQ ID NO:15;phbAdownF:GATGCTCGATGAGTTTTTCTAAATCCTGACAAATTGAACCTGCT,SEQ ID NO:16;phbAdownR:CTGGAAGTCACCACTATCGCA,SEQ ID NO:17;phbAupKF:ATTCATATGAGGTAATTCCTTCATGAGCCATATTCAACGGGAAA,SEQ ID NO:18;phbAdownKR:AGCAGGTTCAATTTGTCAGGATTTAGAAAAACTCATCGAGCATC,SEQ ID NO:19;phbCupF:GGGGATCCGATCATCTTCGACA,SEQ ID NO:20;phbCupR:TTTCCCGTTGAATATGGCTCATGACATTTTTCCTGAAAAACATA,SEQ ID NO:21;phbCdownF:GATGCTCGATGAGTTTTTCTAATCCATCAATCTCAGACCATCGT,SEQ ID NO:22;phbCdownR:TGGGCTTCAGATTAAAGTCACT,SEQ ID NO:23;phbCupKF:TATGTTTTTCAGGAAAAATGTCATGAGCCATATTCAACGGGAAA,SEQ ID NO:24;phbCdownKR:ACGATGGTCTGAGATTGATGGATTAAAAACTCATCGAGCATC,SEQ ID NO:25;upF1:GCGGCGAATTGAAGGCGCACCTCG,SEQ ID NO:26;upR1:TTCCCGTTGAATATGGCTCATCGCCAGCAACAGGCGTTGGCTG\SEQ ID NO:27;downF1:ATGCTCGATGAGTTAGTTAGTTACGTCAGTGCGGTCGGGCTG ID NO:28;downR1:GCCAGTCCAGTTCGACGCAGGGC:SEQ ID NO:29;upKF1:CAGCCAACGCCTGTTGCTGGCGATGAGCCATATTCAACGGGAA,SEQ ID NO:30;downKR1:CGCACTGGAACCCCACCAACTGACTGACTGACTGAQGGAA ID NO:31;phbBupF1:ATTACCGGCGGTGACGCTTCCG:SEQ ID NO:32;phbBupR1:TTTCCCGTTGAATATGGCTCATCGGCATTCGGAAAGCTGCTAGG;SEQ ID NO:33;phbBdownF1:GATGCTCGATGAGTTTTTCTAAAGATTTCCCTTCCTTTTTTTGTC:SEQ ID NO:34;phbBdownR1:ATGCTGAAGAGCACTTTTGAGA\SEQ ID NO:35;phbBupKF1:CCTAGCAGCTTTCCGAATGCCGATGAGCCATATTCAACGGGAAA,SEQ ID NO:36;phbBdownKR1:GACAAAAAAGGAAGGGAAATCTTTAGAAACTCATCGAGCATC,SEQ ID NO:37;phbBupF2:ATGCTGAAGAGCACTTTTGAGA:SEQ ID NO:38;phbBupR2:TTTCCCGTTGAATATGGCTCATAGATTTCCCTTCCTTTTTTGTC:SEQ ID NO:39;phbBdownF2: GATGCCTCGATGAGTTTTTCTAACGGCATTCGGAAAGCTGCTAGG, SEQ ID NO: 40; phbBdownR2: ATTACCGGCGGTGACGCTTCCG, SEQ ID NO: 41; phbBupKF2: GACAAAAAAGGAAGGGAAATCTATGAGCCATATTCAACGGGAAA, SEQ ID NO: 42; phbBdownKR2: CCTAGCAGCTTTCCGAATGCCGTTAGAAAAACTCATCGAGCATC, SEQ ID NO: 43; phbBupF3: ATTACCGGCGGTGACGCTTCCG, SEQ ID NO: 44; phbBupR3: TTTCCCGTTGAATATGGCTCATCGGCATTCGGAAAGCTGCTAGG, SEQ ID NO:45; phbBdownF3:GATGCTCGATGAGTTTTTCTAAAGATTTCCCTTCCTTTTTTGTC, SEQ ID NO: 46; phbBdownR3: ATGCTGAAGAGCACTTTTGAGA, SEQ ID NO: 47; phbBupKF3: CCTAGCAGCTTTCCGAATGCCGATGAGCCATATTCAACGGGAAA, SEQ ID NO: 48; phbBdownKR3: GACAAAAAAGGAAGGGAAATCTTTAGAAAAACTCATCGAGCATC, SEQ ID NO: 49; (3) Construction of linear DNA target fragments: Genomic DNA of wild-type Azotobacter brownii was extracted and used as a template. The primer pairs shown above were then used to amplify Azotobacter brownii. phbB , phbA , phbC Upstream and downstream homologous arms; plasmid pKatAPH3 was extracted and amplified using designed primers. Kan The fragment amplification system is shown in Table 2, and the fragment amplification reaction conditions are shown in Table 3. The obtained target fragments were recovered using a gel extraction kit, and the three target fragments were ligated using homologous recombination enzymes (homological recombination reaction system is shown in Table 4) to obtain linear target fragments. GDMCC No. 1.1487 phbB The nucleotide sequence of the gene is shown in SEQ ID NO.1; GDMCC No. 1.1487 phbA The nucleotide sequence of the gene is shown in SEQ ID NO.2; GDMCC No. 1.1487 phbCThe nucleotide sequence of the gene is shown in SEQ ID NO.3; GDMCC No. 1.1412 phbB The nucleotide sequence of the gene is shown in SEQ ID NO.4; GDMCC No. 1.1413 phbB The nucleotide sequence of the gene is shown in SEQ ID NO.5; GDMCC No. 1.1486 phbB The nucleotide sequence of the gene is shown in SEQ ID NO. 6; Kan The nucleotide sequence of the resistance gene is shown in SEQ ID NO.7; Table 2

[0038] Table 3

[0039] Table 4

[0040] (4) Preparation and transformation of competent cells: Streaking of the wild-type strain onto Burk agar plates (molybdenum and iron removed) and incubation at 30°C for 5–7 days until green fluorescence appears around the colonies (indicating competent cell formation). A loopful of cells is picked and resuspended in 500 μL of P-buffer (4.6 mM K₂HPO₄, 1.5 mM KH₂PO₄). 250–500 ng of the constructed linear DNA fragment is mixed with the competent cell suspension and spotted onto the center of a competent agar plate, incubated overnight at 30°C. The next day, cells are scraped from the plate, resuspended in 1 mL of P-buffer, and serially diluted and plated onto 5 μg / mL kanamycin-resistant Burk agar to obtain single colonies.

[0041] (5) Validation of mutant strains Colony PCR verification: Randomly pick a single colony from the selected plate and perform colony PCR amplification using the verification primers upF and downR located on the outer side of the homologous arm. [[ID=1S3]]ΔphbB The theoretical amplified fragment size of the mutant strain is 2416 bp. Agarose gel electrophoresis results (e.g.) Figure 1 The right figure shows that the successfully selected mutant strains amplified a band that matched the theoretical value, while the wild-type control did not have this band.

[0042] Sequencing verification: The 2416 bp PCR product was sequenced. Sequence alignment results confirmed that... phbB Partial sequences of the gene coding region have been Kan The gene replacement was precise, the homologous recombination event was correct, and no unexpected mutations were introduced.

[0043] Growth and biomass: The mutant strain was inoculated into 1 L of Burk liquid medium and cultured at 220 rpm and 30 °C until the cell mass reached OD. 600 ≈ 1.0. Centrifuge the above bacterial suspensions at 5000 rpm and 4 °C for 10 min, and remove the supernatant. Resuspend the bacterial cells twice in fresh Burk liquid medium and wash twice, centrifuging for 10 min under the same conditions to remove residual medium and bacterial metabolites. Add the washed bacterial suspensions to Burk medium, OD... 600 The temperature was adjusted to 0.3, and the sample was incubated at 220 rpm and 30 °C. Samples were taken every 12 hours, and the absorbance (OD) at 600 nm was measured using a UV-Vis spectrophotometer (Hitachi U-3010, Japan). 600 The growth curve of nitrogen-fixing bacteria was plotted; the number of viable bacteria (colony forming units, CFU) per milliliter of bacterial culture in the sample at 60 h was determined by the dilution plating method, and the bacterial cells at 60 h were collected, dried at 60°C, weighed, and the biomass was determined.

[0044] Nitrogenase activity assay: Nitrogenase activity of *Azotobacter brownings* was determined using the acetylene reduction method. Acetylene gas equivalent to 10% of the headspace volume was injected into a sealed cathode chamber. The chamber was incubated at 220 rpm and 30 °C. Samples were taken every 12 hours, with 250 µL of gas extracted for ethylene concentration analysis by gas chromatography. Sample analysis was performed using an SP gas chromatograph equipped with a flame ionization detector. The analysis was performed using a Model 2100 gas chromatograph (Beijing Beifen Ruili Analytical Instruments Co., Ltd.), with a GDX column packed with a porous polyvinylbenzene stationary phase. 502 column. Ethylene content in gas samples was quantified using ethylene standards after calibration. Protein concentration was determined using the BCA method.

[0045] PHB content determination: Collecting brown nitrogen-fixing bacteria and... phbBThe bacterial cells were dried at 60℃, and 30-40 mg were weighed and added to a mixture of 2 mL chloroform and 2 mL methanol solution (85 wt% methanol, 15 wt% H2SO4, 1 g / L benzoic acid). The mixture was reacted at 100℃ for 3.5 hours. After the reaction solution cooled to room temperature (25℃), 1 mL of water was added for extraction and phase separation. 1 mL of the lower layer (heavy phase) solution was taken for analysis, and 15 mg and 20 mg of pure PHB (Sigma-Aldrich PHB standard) were used as standard samples for the standard curve (Ma et al., 2020). Samples were analyzed using gas chromatography-mass spectrometry (GC-MS, PerkinElmer, Waltham, Massachusetts, USA) equipped with a fused silica capillary column (Elite-5 ms, 30 m × 0.25 mm inner diameter × 0.25 µm). A linear temperature gradient program was used for detection: initial temperature 40 °C held for 1 min, then increased to 120 °C at a rate of 15 °C / min and held for 2 min; then increased to 300 °C at a rate of 10 °C / min and held for 5 min. The injection port temperature was set to 250 °C. Mass spectrometry data were acquired using a 70 eV electron impact ionization source, and the mass-to-charge ratio (m / z) of the scanned mass spectra ranged from 45 to 450. Selected ion monitoring mode was used for the detection of major products and fragment ion analysis (Cho et al., 2022).

[0046] Brown azotobacter and its mutant strains were collected, dried at 60 °C, and 100 mg were weighed and digested using a graphite digester (SH220F, Haineng Scientific Instruments Co., Ltd., China) (each sample required the addition of 0.4 g CuSO4, 6 g K2SO4, and 12 mL H2SO4). The digestion temperature program was set as follows: 160 °C for 60 minutes, 200 °C for 30 minutes, 300 °C for 30 minutes, and 420 °C for 90 minutes. After digestion, the protein content was determined using a fully automated Kjeldahl nitrogen analyzer (K1100, Haineng Scientific Instruments Co., Ltd., China).

[0047] (6) Results Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbB Construction of deletion mutants: to verify phbB The successful construction of the deletion mutant strain involved PCR amplification of randomly selected single clones using specific primers (upF and downR) covering both sides of the gene deletion region. Theoretically predicted... phbB The amplified fragment of the mutant strain was 2416 bp in size, and the agarose gel electrophoresis results were as follows: Figure 1As shown, this is completely consistent with the theoretical value. To further confirm the accuracy of the recombination event, the 2416 bp specific band product was sequenced, and by comparison with the expected sequence, it was confirmed that part of the event was indeed the expected result. phbB The coding region of the gene has been successfully knocked out, and homologous recombination occurred precisely without introducing any unexpected mutations. These results indicate that... phbB The mutant strain has been successfully constructed.

[0048] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbB Growth assay of deletion mutants: to evaluate brown nitrogen-fixing bacteria phbB The effect of gene deletion on bacterial growth was measured in brown azotocin and... phbB The results of viable cell counts showed that the abundance of wild-type *Azotobacter brownii* after 60 h of culture was 0.56 × 10⁻⁶. 8 CFU / mL Figure 2 ), phbB The bacterial abundance was 0.57 × 10⁻⁶. 8 CFU / mL, which can be seen in phbB The gene deletion did not significantly change the number of viable cells per unit, and the above results indicate that... phbB The absence of [something] did not affect cell survival.

[0049] Gas chromatography-mass spectrometry (GC-MS) analysis confirmed that phbB The mutant strain completely lost the ability to synthesize PHB. Figure 3 ).

[0050] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbB Nitrogen fixation capacity assay of deletion mutants: Brown azotobacters and... phbB Nitrogenase activity, results as follows Figure 4 As shown, brown nitrogen-fixing bacteria and phbB There was no significant difference in nitrogenase activity among brown azotocin bacteria. phbB Gene deletion does not affect the nitrogen-fixing ability of brown azotobacters.

[0051] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbB Protein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbB The protein content was determined, and the results are as follows: Figure 5 As shown, phbBThe bacterial protein content was as high as 66.72%, significantly higher than the wild type's 30.23%, representing a 120.71% increase in protein content.

[0052] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbB Determination of carbon source consumption for protein synthesis in deletion mutants: Results are as follows Figure 6 As shown, wild-type and brown nitrogen-fixing bacteria phbB In Burk medium, the total protein content produced by consuming 1 g of carbon source was 33.34 mg and 42.52 mg, respectively. It can be seen that blocking the PHB synthesis pathway not only increases the protein content, but also achieves the production of more protein with the same carbon source consumption.

[0053] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbA Protein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbA The protein content was determined, and the results are as follows: Figure 7 As shown, phbA The bacterial protein content is as high as 65.05%, which is significantly higher than the wild type's 30.23%, representing an increase of 115.18% in protein content.

[0054] Brown nitrogen-fixing bacteria GDMCC No. 1.1487 phbC Protein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbC The protein content was determined, and the results are as follows: Figure 8 As shown, phbC The bacterial protein content is as high as 66.38%, significantly higher than the wild type's 30.23%, with a protein content increase of 119.58%.

[0055] Brown azotocinobacter GDMCC No. 1.1412 phbB Protein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbB The protein content was determined, and the results are as follows: Figure 9 As shown, s The bacterial protein content reached 61.05%, significantly higher than the wild type's 35.12%, representing a 73.83% increase. This successfully enhanced the bacterial protein content of *Azotobacter brownii*.

[0056] Brown azotocinobacter GDMCC No. 1.1413 phbBProtein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbB The protein content was determined, and the results are as follows: phbB As shown, Figure 10 The bacterial protein content is as high as 64.72%, which is significantly higher than the wild type's 31.79%, representing an increase of 103.59% in protein content.

[0057] Brown azotocinobacter GDMCC No. 1.1486 phbB Protein content determination of deletion mutants: The protein content of wild-type and deletion mutants of *Azotobacter brownii* was determined using an automated Kjeldahl nitrogen analyzer. phbB The protein content was determined, and the results are as follows: phbB As shown, Figure 11 The bacterial protein content is as high as 66.19%, which is significantly higher than the wild type's 36.12%, representing an 83.25% increase in protein content.

[0058] The above results indicate that this embodiment successfully constructed the brown azotocin bacterium GDMCC No. 1.1487. phbB , phbA phbB, Gene deletion mutants, and GDMCC Nos. 1.1412, 1.1413, and 1.1486. phbC In gene deletion mutants, the cell protein content of all mutants was significantly increased. This indicates that knocking out a key gene in the PHB synthesis pathway (…) phbB , phbA , phbB This is a universal and effective strategy for increasing the protein content of different brown azotocin strains. By blocking the diversion of carbon sources and reducing power to the energy storage substance PHB, metabolic resources can be directed to the protein synthesis pathway, thereby achieving a substantial increase in the protein content of brown azotocin bacteria. Example 2 This example is used to illustrate brown nitrogen-fixing bacteria. phbC Partial gene deletion mutants ( pvdD Construction and verification of ).

[0059] (1) Strains, culture medium, and primers: The strain was the same as the brown azotocin bacterium with accession number GDMCC No. 1.1417 in Example 1, and the culture medium was the same as in Example 1. The strain... ΔpvdD The full-length gene is 8670 bp. This example utilizes the kanamycin resistance gene ( pvdD Replace the 4311-4781 base region (471 bp in total) within it to construct Kan Partial gene deletion mutant (Δ pvdDThe primers are as shown in Example 1, specifically: primer upF1 / upR1 for amplifying the upstream 800 bp homologous arm of this region; primer downF1 / downR1 for amplifying the downstream 800 bp homologous arm; and primers for amplifying... pvdD The primers for the resistance gene are upKF1 / downKR1.

[0060] (2) Construction and transformation of linear DNA target fragments: The method is the same as in Example 1. Using genomic DNA as a template, homologous arm fragments on both sides of the target region are amplified using primer pairs upF1 / upR1 and downF1 / downR1, respectively. Using plasmid pKatAPH3 as a template, the homologous arm fragments are amplified using primer pair upKF1 / downKR1. Kan The fragments were homologously recombinated to obtain linear DNA fragments. Using the same competent cell preparation method and transformation procedure as in Example 1, the linear fragments were transformed into wild-type strains and screened on Burk plates containing kanamycin.

[0061] (3) Mutant strain verification: Colony PCR verification: Transformants were amplified by PCR using verification primers upF1 and downR1. Theoretically, the wild-type amplified fragment was predicted to be 2070 bp, while... Kan The mutant strain was inserted [[ID=22o]]ΔpvdD The gene fragment size should be 2416 bp. Agarose gel electrophoresis results (e.g.) Kan As shown in the image, clones with amplified band sizes matching the theoretical values ​​for the mutant strain were successfully obtained. Sequencing verification: Sequencing of the 2416 bp PCR product confirmed... Figure 12 The target region of the gene has been pvdD Precise gene replacement.

[0062] Phenotypic verification: Macroscopic observation: After culturing in Burk liquid medium for 60 hours, the culture supernatant of the wild-type strain showed yellow-green fluorescence, while Kan The culture supernatant of the mutant strain was colorless and non-fluorescent (e.g. ΔpvdD (As shown). Ultraviolet spectroscopy analysis: Ultraviolet-visible spectral scanning was performed on the culture supernatant, such as... Figure 13 As shown, the wild-type supernatant has a characteristic absorption peak at 380 nm, while [[ID=zz7]]Figure 14 The mutant strain supernatant showed no absorption peak at this wavelength. This indicates that... ΔpvdD The mutant strain lost the ability to synthesize this fluorescent siderophore.

[0063] (4) Performance testing of mutant strains: growth and biomass: growth curves (e.g.) ΔpvdD (as shown) and cell dry weight after 60 hours of culture (as shown) Figure 15 The measurement results (as shown) indicate that Figure 16 The mutant strain showed no significant difference from the wild type, indicating that... ΔpvdDPartial gene deletion has no effect on bacterial growth. Nitrogenase activity: Results from the acetylene reduction method showed that... pvdD The mutant strain showed no significant difference in nitrogenase activity compared to the wild type (e.g. ΔpvdD As shown in the figure, this indicates that the mutation does not affect nitrogen fixation capacity. Protein content: Kjeldahl nitrogen determination showed that... Figure 17 The protein content of the mutant strain was 29.36%, which was not significantly different from the wild type's 30.23% (e.g., ΔpvdD (As shown). The above results indicate that the system constructed in this embodiment... Figure 18 Mutants with partial gene deletions ( pvdD ΔpvdD This mutant strain lost the ability to synthesize fluorescent siderophores, but its growth, nitrogen fixation ability, and cell protein content did not change significantly. Therefore, deleting the fluorescent siderophore synthesis pathway alone did not increase the cell protein content of *Azotobacter brownii* under the conditions of this embodiment.

[0064] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0065] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0066] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for increasing the protein content of brown azotocin bacteria, characterized in that, The method includes inactivating the PHB synthesis pathway in the brown azotocin bacteria.

2. The method according to claim 1, wherein, The brown azotoxin bacteria mentioned are those with preservation numbers GDMCC No.1.1487, GDMCC No.1.1412, GDMCC No.1.1413, and GDMCC No.1.1486.

3. The method according to claim 1, wherein, The PHB synthesis pathway in the inactivated brown azotocin includes at least one of gene editing, gene silencing, and enzyme activity inhibition.

4. The method according to claim 3, wherein, The gene editing includes knocking out, mutating, or replacing key genes in the PHB synthesis pathway; The key genes include phbA , phbB , phbC At least one of the genes.

5. The method according to claim 4, wherein, The method includes removing the brown azotocin from the bacteria. phbA, phbB or phbC The gene was replaced with a resistance gene; The resistance gene is Kan Gene.

6. The use of the method according to any one of claims 1-5 in the production of single-cell proteins.

7. The use of the method according to any one of claims 1-5 in the preparation of feed protein.

8. The application of the method according to any one of claims 1-5 in the preparation of food ingredients.

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