Protein-producing pseudomonas and application thereof

Abstract

The invention discloses a pseudomonas proteinogenes strain and an application thereof, and relates to the field of microorganisms, in particular to the pseudomonas proteinogenes strain and the application thereof. The invention aims to solve the problem that the single-cell protein producing bacteria can only produce the single-cell protein under the illumination condition, and the energy consumption is large. The strain is pseudomonas proteinogenes Y24-6 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is December 16, 2020, and the preservation number is CGMCC No.21380. The invention further discloses a preparation method of the pseudomonas proteinogenes Y24-6. The bacillus subtilis is a new bacterium and can be used for producing single-cell protein through fermentation. By analyzing the amino acid components of the produced single-cell protein, 16 amino acids are detected in total. The method is used for fixing the carbon dioxide single-cell protein.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a protein-producing Pseudomonas bacterium and its application. Background Art

[0002] Single-cell protein (microbial protein) is protein produced by microorganisms, including bacterial protein, fungal protein, etc. Yeasts, bacteria, and algae can all be used to produce single-cell protein. With the increasingly prominent problems such as population growth, global warming, and food security, there is an urgent need for new sustainable protein sources to reduce pollution to the ecological environment. Therefore, the technology of producing single-cell protein with low cost and sustainability by biological fixation and sequestration of CO2 has broad application prospects.

[0003] Both autotrophic microorganisms and heterotrophic microorganisms can fix CO2. Autotrophic microorganisms can use CO2 as their sole carbon source. For example, algae can photosynthetically fix CO2 to directly produce high-value chemicals such as biodiesel and biofertilizer. Compared with autotrophic microorganisms, heterotrophic microorganisms can produce high-value chemicals at a higher production rate under dark conditions. Heterotrophic microorganisms obtain energy by decomposing organic substrates and achieve net fixation of CO2 when producing certain specific products (such as malic acid and succinic acid). In recent years, it has been found that facultative autotrophic microorganisms can use organic matter as a carbon source or CO2 as the sole carbon source. For example, a strain of aerobic-facultative autotrophic denitrifying bacterium Pseudomonas koreensis Y5-11 disclosed in patent CN109517770A can use CO2 as the sole carbon source to reduce nitrate.

[0004] Currently, the common bacteria producing single-cell protein are purple photosynthetic bacteria. Purple photosynthetic bacteria can fix CO2 under anaerobic conditions and use light to synthesize single-cell protein; they can also use light as an energy source and organic carbon as a carbon source for photoheterotrophic metabolism to synthesize single-cell protein. Therefore, the production of single-cell protein by purple photosynthetic bacteria requires an artificial lighting system, which consumes a large amount of energy and has high operating costs. Summary of the Invention

[0005] The present invention aims to solve the problem that bacteria producing single-cell protein need to carry out under light conditions and consume a large amount of energy, and provides a protein-producing Pseudomonas bacterium and its application.

[0006] The protein-producing Pseudomonas bacterium of the present invention is Pseudomonas proteinensis Y24-6, which is deposited in the China General Microbiological Culture Collection Center. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 16, 2020, and the deposit number is CGMCC No. 21380.

[0007] Morphological characteristics of Pseudomonas proteinensis Y24-6 of the present invention: The cells are rod-shaped, 1.24 μm to 1.88 μm in length and 0.76 μm to 0.85 μm in width, with flagella; Gram-negative, without spores; The colonies are round dots, milky white, semi-transparent, raised, and easy to pick up.

[0008] Physiological and biochemical characteristics of Pseudomonas proteinensis Y24-6 of the present invention: It is an obligate aerobe and can grow under the conditions of 4 - 35 °C and pH 6 - 8; The optimum growth temperature is 30 °C, and the optimum growth pH value is 6.8. The available carbon sources include β-D-glucose, 1% sodium lactate, D-mannitol, L-alanine, L-aspartic acid, L-glutamic acid, L-serine, L-lactic acid, quinaldine sulfate, D-gluconic acid, glucuronamide, quinic acid, mucic acid, α-oxo-glutamic acid, citric acid, L-malic acid, and γ-aminobutyric acid.

[0009] Results of molecular biological identification of Pseudomonas proteinensis Y24-6 of the present invention: Through 16S rDNA sequence alignment analysis, the similarity with its closely related species Pseudomonas migulae is 99.42%. The G + C content of Pseudomonas proteinensis Y24-6 is 59.68%, which is higher than the G + C content (58%) of Pseudomonas migulae. Through combining the cell morphological characteristics, growth conditions, and physiological and biochemical identification results, it is determined that Pseudomonas proteinensis Y24-6 is a new species of bacteria.

[0010] Pseudomonas proteinensis Y24-6 of the present invention is used for fermentative production of single-cell protein.

[0011] Furthermore, the fermentation conditions are 20 - 30 °C and 180 rpm.

[0012] Furthermore, the fermentation uses Fe 2+ as an electron donor.

[0013] Furthermore, the C / N ratio of the medium used for fermentation is 0.24 - 0.32.

[0014] Advantages of the present invention:

[0015] The Pseudomonas proteinensis Y24-6 screened in the present invention is a new species of bacteria that can ferment and produce single-cell protein. By analyzing the amino acid components of the produced single-cell protein, 16 amino acids were detected in total. During the process of fixing carbon dioxide to produce single-cell protein by the Pseudomonas proteinensis Y24-6 of the present invention, complex conditions such as light are not required.

[0016] During the fermentation process, when Fe 2+ is used as the electron donor, the protein yield of Pseudomonas proteinensis Y24-6 is relatively high. When the C / N ratio of the culture medium is 0.24, the protein production of Pseudomonas proteinensis Y24-6 is the highest, which is 0.375 g protein / g dry cell weight of the bacteria.

[0017] The Pseudomonas proteinensis Y24-6 of the present invention can fix carbon dioxide to produce single-cell protein. Brief Description of the Drawings

[0018] Figure 1 shows the cell morphology of Pseudomonas proteinensis Y24-6 of the present invention;

[0019] Figure 2 shows the colony morphology of Pseudomonas proteinensis Y24-6 of the present invention;

[0020] Figure 3 shows the amino acid components of the single-cell protein produced by Pseudomonas proteinensis Y24-6 detected at a wavelength of 570 nm;

[0021] Figure 4 shows the amino acid components of the single-cell protein produced by Pseudomonas proteinensis Y24-6 detected at a wavelength of 440 nm. Detailed Embodiments

[0022] The following will give a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0023] Example 1:

[0024] The Pseudomonas proteinensis Y24-6 in this example is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 16, 2020, and the deposit number is CGMCC No. 21380.

[0025] The method for obtaining the Pseudomonas proteinensis Y24-6 in this example is as follows:

[0026] Using the low-temperature groundwater (4 - 6°C) in the Harbin area as the screening source, add the screening solution and culture at 4 - 6°C for more than 3 days. After gradient dilution, perform streak separation at 4 - 6°C, and select typical single colonies for purification culture, which is the strain Y24-6 of this example.

[0027] The components of the screening solution are as follows: nitrogen source 0.3 - 0.5 g / L, carbon source 0.1 - 1 g / L, iron salt 0.01 - 0.1 g / L, NaCl 0.1 - 0.5 g / L, MgSO4·7H2O 0.05 - 0.1 g / L, MnSO4 0.01 - 0.1 g / L, CaCl2 0.1 g / L, Na2HPO4 0.1 - 0.2 g / L, pH value 7.0. Among them, the carbon source is carbon dioxide, Na2CO3, NaHCO3, ethanol or sodium acetate, the nitrogen source is NaNO3 or KNO3, and the iron salt is (NH4)2Fe(SO4)2·6H2O or FeSO4.

[0028] Example 2: Identification of strain Y24-6

[0029] In this example, the physiological and biochemical identification of strain Y24-6 was carried out with reference to the eighth edition of Bergey's Manual of Determinative Bacteriology and the Manual of Systematic Identification of Common Bacteria:

[0030] The morphological characteristics of the strain are: rod-shaped, as Figure 1 shown, with a length of 1.24 μm - 1.88 μm and a width of 0.76 μm - 0.85 μm, and having flagella; Gram-negative staining, without spores; the colony is round dot-shaped, milky white, semi-transparent, protruding, and easy to pick up, as Figure 2 shown.

[0031] The physiological and biochemical characteristics of the strain are: obligate aerobe, which can grow at 4 - 35°C and pH 6 - 8; the optimal growth temperature is 30°C, and the optimal growth pH value is 6.8. The available carbon sources include β-D-glucose, 1% sodium lactate, D-mannitol, L-alanine, L-aspartic acid, L-glutamic acid, L-serine, L-lactic acid, quinine tetra sulfate, D-gluconic acid, glucuronamide, quinic acid, mucic acid, α-oxidized-glutamic acid, citric acid, L-malic acid, and γ-aminobutyric acid.

[0032] For the molecular identification of strain Y24-6, after extracting the genomic DNA of strain Y24-6, 16S rDNA sequence amplification was carried out. The PCR reaction primers used for amplification were universal primers. The PCR reaction system was as follows: 50-100 ng of template DNA, 25 μL of Taq enzyme mixture, 0.5 μL of forward primer, 0.5 μL of reverse primer, and sterile deionized water was added to make up to 50 μL. PCR amplification conditions: pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 1 min, amplification at 50-55°C for 1 min, extension at 72°C for 1.5 min. After 30 cycles, extension at 72°C for 5 min.

[0033] Forward primer 5’-GAGCGGATAACAATTTCACACAGG-3’;

[0034] Reverse primer 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’.

[0035] The fragment obtained by PCR was used for sequencing, and a 16S rDNA sequence of 1404 bp was obtained. The 16S rDNA sequence is shown as SEQ ID NO: 1 in the sequence listing.

[0036] Its similarity with the related species Pseudomonas migulae was 99.42%. Although the similarity was high, the G+C content of Pseudomonas proteinensis Y24-6 was 59.68%, which was significantly higher than that of Pseudomonas migulae (58%). By combining the cell morphology characteristics, growth conditions, and physiological and biochemical identification results, it was determined that the strain Y24-6 in this example was a new species of bacteria, named Pseudomonas proteinensis Y24-6.

[0037] Example 3: Analysis of the single-cell protein content and amino acid composition of Pseudomonas proteinensis Y24-6

[0038] The fresh bacterial liquid cultured for 16-24 h (cell concentration 10 9Centrifuge at 6000 rpm for 5 min. Take the precipitated bacteria after centrifugation. Add 1.5 mL of sterile deionized water to the centrifuge tube and pipette the bacteria evenly. Repeatedly freeze-thaw 4 times in liquid nitrogen and a 37°C forced-air drying oven in sequence to break the cell membrane of strain Y24-6, and then centrifuge at 12000 rpm for 30 min. Take 1 mL of the supernatant and transfer it to a clean 1.5 mL centrifuge tube. Add 0.1 mL of 72% trichloroacetic acid solution, mix well by shaking, and then let it stand and shake well for 10 min. Then centrifuge at 5000 rpm for 5 min and discard the supernatant. Take the precipitate and add 1.5 mL of 1×PBS buffer (Shanghai Aladdin Biochemical Technology Co., Ltd.) to dissolve the precipitate. Then measure the protein content according to the operation steps of the BCA Protein Concentration Assay Kit (Shanghai Sangon Biotech Co., Ltd.). The results show that the single-cell protein content of strain Y24-6 is 0.320 ± 0.005 g protein / g dry cell weight of bacteria.

[0039] Analyze the amino acid composition of the single-cell protein using an amino acid analyzer. The results are as Figure 3 and Figure 4 shown. The amino acid components detected at a wavelength of 570 nm include: 1. Aspartic acid (Asp), 2. Threonine (Thr), 3. Serine (Ser), 4. Glutamic acid (Glu), 5. Glycine (Gly), 6. Alanine (Ala), 7. Valine (Val), 8. Methionine (Met), 9. Isoleucine (Ile), 10. Leucine (Leu), 11. Tyrosine (Tyr), 12. Phenylalanine (Phe), 13. Lysine (Lys), 15. Histidine (His), 16. Arginine (Arg); the amino acid detected at a wavelength of 440 nm is 17. Proline (Pro). A total of 16 amino acids are detected. The 14th peak in the figure is NH3.

[0040] Example 4: Optimal protein production conditions test for Pseudomonas proteinensis Y24-6 to produce single-cell protein by fixing carbon dioxide

[0041] Using Fe(SO4)2·7H2O and Na2S2O3 as electron donors respectively, set the concentration of Fe 2+ to 50 mg / L and the concentration of S to 50 mg / L. Using CO2 / NaHCO3 as the carbon source, adjust the pH value balance, and set the C / N of the medium to 0, 0.24, 0.32, 0.4, 0.48, 0.56, and 0.64. Cultivate at 20°C and 180 rpm for 24 h, and measure the protein production. The results are shown in Table 1.

[0042] Compared with Na2S2O3, when Fe(SO4)2·7H2O was used as the electron donor, the protein production of strain Y24-6 was higher. Moreover, when the inorganic C / N ratio was 0.24, the protein production of strain Y24-6 was the highest, reaching 0.375 g protein / g dry cell weight of the bacteria. Therefore, when Pseudomonas proteinensis Y24-6 fixes carbon dioxide to produce single-cell protein, Fe 2+ should be used as the electron donor, and the optimal condition is an inorganic C / N ratio of 0.24.

[0043] Table 1 Protein production of strain Y24-6 under different electron donors and inorganic C / N ratios (g protein / g dry cell weight of the bacteria)

[0044]

Claims

1. A protein-producing Pseudomonas, characterized in that The bacterium is Pseudomonas proteinensis Y24-6, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 16, 2020, and the deposit number is CGMCC No. 21380.

2. The application of Pseudomonas proteolytica according to claim 1, characterized in that The Pseudomonas proteinensis is used for fermentative production of single cell protein.

3. The application according to claim 2, wherein The fermentation conditions are 20-30 °C and 180 rpm.

4. The application according to claim 2 or 3, characterized in that The fermentation uses Fe 2+ as an electron donor.

5. The application according to claim 2, wherein The C / N ratio of the culture medium used for fermentation is 0.24-0.32.

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