Pseudoromonas strain and application thereof

Abstract

The invention discloses a pseudoromonas sp. Strain and application thereof, the strain is a new species in pseudoromonas sp., and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No. 1.14501. The invention further discloses a preparation method of the pseudoromonas sp. The strain E2-1-a4 obtained through separation is a new species which is different from those of known species of Falsiroseomonas, and the strain E2-1-a4 can be used for producing poly (beta-hydroxybutyric acid) (PHB). The produced PHB has very wide development and application prospects in medicine, agriculture and packaging materials.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Pseudoromonas strain and application thereof in synthesizing PHB. Background Art

[0002] Roseomonas was first proposed by Rihs et al. in 1993, and Falsiroseomonas was separated from Roseomonas by Rai et al. in 2021. It belongs to Pseudomonadati, Pseudomonadota, Alphaproteobacteria, Rhodospirillales, Acetobacteraceae. The main characteristics of Falsiroseomonas are: aerobic, Gram-negative, coccobacillus; the main respiratory quinone is ubiquinone Q-10, and the polar lipids are mainly diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and aminophospholipids (AL).

[0003] Falsiroseomonas is widely distributed and can survive in a variety of environments, including water cooling systems, soil, water bodies, etc. For example, Falsiroseomonas tokyonensis was isolated from a cooling tower biofilm sample in Tokyo, Japan, and Falsiroseomonas frigidaquae was first isolated from a water cooling system in South Korea.

[0004] This genus of bacteria has diverse metabolic capabilities and can use a variety of carbon and nitrogen sources for growth and metabolism. Some strains can use simple organic compounds such as glucose and sucrose as carbon sources, and can also use ammonium salts, nitrates, etc. as nitrogen sources. When the carbon source in the environment is sufficient, but other nutrients such as nitrogen and phosphorus sources are relatively scarce, Falsiroseomonas will convert the excess carbon source into PHB and store it. In the case of insufficient carbon source, PHB can be decomposed into acetyl coenzyme A and other substances that enter the tricarboxylic acid cycle, providing cells with energy and carbon skeletons to maintain cell growth, reproduction, metabolism and other life activities. In addition, in high salt or other environments that cause changes in extracellular osmotic pressure, Falsiroseomonas synthesizes and accumulates PHB to balance the osmotic pressure inside and outside the cell, prevent cells from being damaged due to osmotic pressure imbalance, and ensure the normal physiological function of cells.

[0005] Poly-β-hydroxybutyrate (PHB), whose monomer is (R)-3-hydroxybutyric acid, is connected by ester bonds to form linear polymers. It has the characteristics of plastics, such as thermoplasticity and ductility, can be spun and pressed into films, and is also biodegradable, and is widely used in various fields. For example, it has great potential in medical implant materials, and can be used as raw materials for surgical sutures, patch materials, and various stents used in surgical operations. It can also be made into gels, microspheres, nanoparticles, etc. to load drugs; in agriculture, PHB can be made into farmland film due to its biodegradability to reduce white pollution, and can also be used as a carrier of fertilizers and pesticides to slowly release fertilizers and pesticides; in packaging materials, PHB also has good barrier properties and is used to make packaging for volatile foods such as tea and spices.

[0006] At present, the methods for synthesizing PHB are mainly gene synthesis, chemical synthesis and microbial synthesis. Among them, gene synthesis is relatively complex, chemical synthesis is more expensive and has by-products that affect the quality of PHB. In contrast, microbial synthesis can use renewable resources as raw materials, such as plant straw hydrolysate, waste molasses, etc., and the synthesis process is mild, without extreme conditions, which can reduce energy consumption and equipment requirements; and the synthesized PHB has good biodegradability and can be decomposed by microorganisms in the natural environment, reducing pollution to the environment. Therefore, the production of PHB using microbial synthesis has broad application prospects. Summary of the invention

[0007] One of the objectives of the present invention is to provide a Pseudomonas sp. strain E2-1-a4.

[0008] The Pseudoroseomonas strain E2-1-a4 provided by the present invention is isolated from the soil on the shore of Longmu Co Lake in Ali Prefecture, Tibet Autonomous Region, and the strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.1.14501 and a deposit date of February 19, 2025. After bacterial strain identification, it was found that the strain is a new species of the genus Falsiroseomonas.

[0009] The study found that the strain E2-1-a4 can utilize α-D-glucose, D-fucose, D-fructose-6-phosphate, L-histidine, D-galacturonic acid, D-galactonolactone, D-gluconic acid, D-glucuronic acid, glucosamide, D-glucaric acid, L-lactic acid, citric acid, α-ketoglutaric acid, D-malic acid, L-malic acid, bromosuccinic acid, β-hydroxy-DL-butyric acid, and formic acid as the sole carbon source, and has a broad spectrum of substrates.

[0010] In addition, according to physiological experiments, strain E2-1-a4 can grow at a temperature of 15-30°C, a pH of 6.0-10.0, and a NaCl concentration of 0%-2.0%. The optimal growth conditions of strain E2-1-a4 are 26°C, a pH of 9.0, and a NaCl concentration of 0.5%.

[0011] The second object of the present invention is to provide a fermentation broth and a liquid inoculum prepared from the above-mentioned Pseudoromonas strain and a preparation method thereof, the method comprising:

[0012] 1) Strain activation: Select the strain and streak it onto R2A solid culture medium, place it in a constant temperature incubator, and culture it at 26°C for 48 hours.

[0013] 2) Preparation of seed solution: Pick activated bacteria and inoculate them into a large test tube containing seed culture medium, seal it with a rubber stopper, place it on a shaker at 26°C and shake at 150 rpm for 16-24 hours to obtain seed solution.

[0014] 3) Liquid fermentation: The seed liquid and seed culture medium are then expanded with an inoculation amount of 5-10% by volume, and the culture conditions are 26°C and 150rpm; the culture obtained after 72 hours of culture is the fermentation liquid; the fermentation liquid is centrifuged at 8000rpm for 15 minutes to collect the bacteria.

[0015] Among them, the seed culture medium is R2A liquid culture medium, and the formula of the R2A liquid culture medium is: 0.5g yeast extract, 0.5g peptone, 0.5g tyrosine, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.05g potassium dihydrogen phosphate, distilled water is added to make the volume to 1000mL, and sterilized at 121°C for 20min; the formula of the R2A solid culture medium is 15.0g agar added to the R2A liquid culture medium.

[0016] The third object of the present invention is to provide the use of the above-mentioned Pseudoromonas strain and the fermentation broth and liquid inoculant prepared by the strain in the synthesis of PHB.

[0017] Through transmission electron microscopy observation, it was found that the strain E2-1-a4 contained transparent particles in the ultrathin section, and the particles were identified as PHB.

[0018] Then, the extract was carried out according to the PHB extraction method, and it was found that PHB could be extracted. The specific extraction method includes the following steps:

[0019] 1) Prepare the fermentation broth and collect the cells according to the method described above.

[0020] 2) After fixing the bacteria with a fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde, ultrathin sections were prepared and a transmission electron microscope was used to observe whether the inside of the bacteria contained transparent particles; the precipitated bacteria were freeze-dried and weighed, and 30 mL of chloroform and 30 mL of sodium hypochlorite aqueous solution (30%, v / v) were added per gram of dry cell weight.

[0021] 3) The above mixture was placed in a shaker (150 rpm, 30° C.) and shaken for 90 min.

[0022] 4) After centrifugation (3000 g, 15 min), the mixture was separated into three phases: the upper layer was sodium hypochlorite solution, the middle layer was cell fragments without PHB, and the lower layer was chloroform containing PHB.

[0023] 5) Use a pipette to suck up the upper solution, and then filter it through filter paper to obtain the chloroform phase.

[0024] 6) Finally, add ten times the volume of pre-cooled methanol to the chloroform phase to precipitate PHB.

[0025] Advantages of the present invention:

[0026] The pseudoroseomonas E2-1-a4 of the invention is separated from soil on the shore of Longmu Co Salt Lake in Ali area of ​​Tibet Autonomous Region, and the environment is not polluted, and the invention has no ecological toxicity, high safety and strong stability.

[0027] The pseudoroseomonas E2-1-a4 of the present invention is identified as a new species of the genus Falsiroseomonas through 16S rRNA gene sequence analysis, genome analysis, phylogenetics and multiple taxonomic analyses.

[0028] The pseudoroseomonas E2-1-a4 of the invention has simple culture, short cycle and strong stability, and can produce PHB. The produced PHB has very broad development and application prospects in medicine, agriculture and packaging materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Morphology of strain E2-1-a4 under transmission electron microscopy.

[0030] Figure 2 : Phylogenetic tree of 16S rRNA gene of strain E2-1-a4.

[0031] Figure 3 : Ultrathin section image of strain E2-1-a4 under transmission electron microscope. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0033] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0034] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0035] Example 1 Isolation and identification of Pseudoromonas strain E2-1-a4

[0036] a. Soil sample collection: Soil was collected from the shore of Longmu Co Lake in Ali Prefecture, Tibet Autonomous Region, placed at 4°C and brought back to the laboratory for strain isolation using the plate dilution coating method.

[0037] b. The culture medium for isolating the strain is R2A solid culture medium: 0.5 g yeast extract, 0.5 g peptone, 0.5 g tyrosine, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.05 g potassium dihydrogen phosphate, 15 g agar, dilute to 1000 mL with distilled water, and sterilize at 121°C for 20 min.

[0038] c. Separation by plate dilution and coating method: Mix the collected soil samples thoroughly, weigh 10g, put into a triangular flask with appropriate amount of glass beads and 90mL sterile water, place the triangular flask on a shaker, shake for 20min, let stand for 5min, dilute in sequence by 10-fold gradient dilution method, take 10 -4 , 10 -5 , 10 -6 200 μL of soil dilution was evenly spread on R2A solid plates. Three plates were spread for each dilution, inverted and cultured in a 20°C constant temperature incubator for 14 days, and single colonies with different colony morphology were selected for further streaking purification. The purified strains were transferred to R2A slant medium and stored at 4°C for later use.

[0039] d. Identification of strains: After streaking, the purified strain E2-1-a4 was streaked on an R2A plate and the colony morphology was observed: the colonies were round, pink, smooth, opaque, and had neat edges. The cell morphology was observed under an electron transmission microscope and the cells were spherical ( Figure 1). The PCR amplicon was sequenced using 16S rRNA universal primers 27F and 1492R, and the resulting sequence is shown in SEQ ID NO.1. By comparing the 16S rRNA sequences of known strains in the Genbank database, it was found that the 16S rRNA gene sequence of strain E2-1-a4 was the most similar to that of Falsiroseomonas frigidaquae CW67, with a value of 98.60%. According to the Bergey Manual of Bacterial Identification and multiple taxonomic methods, the phenotypic and physicochemical characteristics of the strains were analyzed, and the phylogenetic tree analysis was combined with the 16S rRNA gene sequence ( Figure 2 ) and genome sequencing analysis, it was determined that the E2-1-a4 strain was a new species of the genus Falsiroseomonas (Falsiroseomonas sp.nov.). The strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.1.14501 and the deposit date of February 19, 2025.

[0040] Wherein, the sequence of SEQ ID NO.1 is as follows:

[0041]

[0042]

[0043] After studying the strain, it was found that strain E2-1-a4 can use α-D-glucose, D-fucose, D-fructose-6-phosphate, L-histidine, D-galacturonic acid, D-galactonolactone, D-gluconic acid, D-glucuronic acid, glucosamide, D-glucaric acid, L-lactic acid, citric acid, α-ketoglutaric acid, D-malic acid, L-malic acid, bromosuccinic acid, β-hydroxy-DL-butyric acid, and formic acid as the sole carbon source. In addition, according to physiological experiments, strain E2-1-a4 can grow at a temperature of 15-30°C, a pH of 6.0-10.0, and a NaCl concentration of 0%-2.0%. The optimal growth conditions for strain E2-1-a4 are 26°C, a pH of 9.0, and a NaCl concentration of 0.5%.

[0044] Example 2 Preparation of Fermentation Broth of Pseudoromonas strain E2-1-a4

[0045] a. Strain activation: Pick the strain and streak it onto R2A solid medium (0.5 g yeast extract, 0.5 g peptone, 0.5 g tyrosine, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.05 g potassium dihydrogen phosphate, 15 g agar, dilute to 1000 mL with distilled water, pH 7.2-7.4, sterilize at 121°C for 20 min), and culture in a constant temperature incubator at 26°C for 48 h.

[0046] b. Preparation of seed solution: Pick the activated strain and inoculate it into R2A liquid medium (its formula and preparation are the same as the above R2A solid medium, without adding agar), and culture it at 26°C and 150rpm for 16-24 hours to obtain liquid seeds.

[0047] c. Liquid fermentation: According to the liquid seeds and fermentation medium (formulation and preparation are the same as the above R2A liquid medium) with a volume ratio of 5-10% inoculation amount for expansion culture, the culture conditions are 26 ° C, 150 rpm; the culture obtained by shaking culture for 72h is the fermentation broth.

[0048] Example 3 Transmission electron microscopy observation of Pseudoromonas strain E2-1-a4 synthesizing PHB

[0049] a. The fermentation broth in Example 2 was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the precipitated bacteria were collected.

[0050] b. Take 20N30 μL of the precipitated bacteria in a 1.5mL EP tube, fix it with 0.2M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8), 2.5% glutaraldehyde, and 1% paraformaldehyde at room temperature for 30 minutes, and then place it at 4℃ for 1.5 hours (shake the liquid several times during fixation to ensure that the liquid is fully mixed). Finally, store it at 4℃.

[0051] c. Centrifuge the fixative in b (6000 rpm, 3 min) to collect the bacteria; mix the warm, non-condensing agar and the bacteria, and after cooling, cut off the excess agar, leaving the agar mass containing the bacteria; then rinse with 0.1 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH = 8) 5 times, 5 min each time.

[0052] d. After rinsing, soak the agar block containing the bacteria in osmium acid solution (this process is carried out on ice) and observe whether the color of the agar block turns dark brown. After the color changes, observe for another 10 minutes. If the color does not change, remove the osmium acid solution. Rinse with 0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8) for 3 times, 5 minutes each time.

[0053] e. Dehydration was performed using 30%, 50%, 70%, 85%, 95%, and 100% ethanol solutions. 1 ml of the above ethanol solutions was added to the sample after treatment d and the sample was soaked for 3 min. Except for the 100% anhydrous ethanol dehydration which was performed at room temperature, the rest were performed on ice. The 100% anhydrous ethanol dehydration operation needed to be repeated 5 times.

[0054] f. Use a mixture of anhydrous ethanol and resin as a penetrant, with a ratio of 1:1 (anhydrous ethanol: resin). Add the prepared penetrant to the sample after dehydration in e, with an amount that is appropriate to immerse the sample, and let it stand for 1.5 hours. After that, suck out the penetrant, add pure resin, and place it in a desiccator overnight.

[0055] g. After the sample is prepared, it is ultra-thinly sliced ​​and observed under a transmission electron microscope.

[0056] The results showed that in the ultrathin section of strain E2-1-a4 ( Figure 3 ), the cells are generally spherical, with many spherical transparent particles scattered inside the cells and the transparent particles are of different sizes, which is consistent with the known imaging characteristics of PHB under electron microscope. Because PHB has a low electron density, it appears as a transparent area in the image. In addition, the transparent particles in the figure are relatively regular spherical particles, and there is no obvious pattern in their distribution inside the cells, which is also consistent with the characteristics of PHB being stored in the form of particles inside the cells.

[0057] In order to further confirm from a genomic perspective that these transparent particles are PHB, the whole genome of strain E2-1-a4 was sequenced. Through bioinformatics analysis, the key genes phaA, phbB and phaC related to PHB synthesis were successfully identified in its genome. Among them, the phaA gene encodes β-ketothiolase, which is responsible for converting acetyl-CoA into acetoacetyl-CoA; the phbB gene encodes NADPH-dependent acetoacetyl-CoA reductase, which can catalyze the conversion of acetoacetyl-CoA into β-hydroxybutyryl-CoA; the phaC gene encodes polyhydroxy fatty acid synthase, which can catalyze the polymerization of β-hydroxybutyryl-CoA to form PHB. The presence of these key genes provides strong genetic evidence for the ability of strain E2-1-a4 to synthesize PHB, thereby further confirming that the transparent particles observed in the ultrathin section are PHB.

[0058] Among them, phaA is located in Scaffold 16, with a size of 2973bp; phbB is located in Scaffold 9, with a size of 723bp; phaC is located in Scaffold 16, with a size of 345bp.

[0059] The phaA sequence (SEQ ID NO. 2) is as follows:

[0060]

[0061]

[0062] The phbB sequence (SEQ ID NO.3) is as follows:

[0063] The phaC sequence (SEQ ID NO.4) is as follows:

[0064]

[0065]

[0066] Example 4 Extraction of PHB synthesized by Pseudoromonas strain E2-1-a4

[0067] According to the extraction steps of PHB, PHB in the fermentation broth is extracted. The specific steps are as follows:

[0068] a. The fermentation broth in Example 2 was centrifuged at 8000 rpm for 15 min to collect the precipitated bacterial cells. The precipitated bacterial cells were freeze-dried and weighed, and 30 mL of chloroform and 30 mL of sodium hypochlorite aqueous solution (30%, v / v) were added per gram of dry cell weight.

[0069] b. Place the above mixture in a shaker (150 rpm, 30°C) and shake for 90 min.

[0070] c. After centrifugation (3000g, 15min), the mixture was separated into three phases: the upper layer was sodium hypochlorite solution, the middle layer was cell fragments without PHB, and the lower layer was chloroform containing PHB.

[0071] d. Use a pipette tip to suck up the upper solution, and then filter it through filter paper to obtain the chloroform phase.

[0072] e. Finally, add ten volumes of pre-cooled methanol to the chloroform phase to precipitate PHB.

[0073] It has been verified that PHB can be extracted from the fermentation broth of Pseudoromonas strain E2-1-a4 using the above method.

[0074] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A strain of Pseudoromonas, characterized in that The strain is Falsiroseomonas sp. strain E2-1-a4, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No.1.14501.

2. The Pseudoromonas strain according to claim 1, characterized in that The 16S rRNA gene sequence of the strain is shown in SEQ ID NO.

1.

3. The Pseudoromonas strain according to claim 1, characterized in that The strain E2-1-a4 can grow under the conditions of a temperature of 15-30° C., a pH value of 6.0-10.0, and a NaCl concentration of 0%-2.0%.

4. The Pseudoromonas strain according to claim 3, characterized in that The optimal growth conditions of the strain E2-1-a4 are 26° C., pH 9.0, and NaCl concentration 0.5%.

5. The fermentation liquid and liquid bacterial agent prepared by the Pseudoromonas strain according to claim 1.

6. A method for preparing the fermentation liquid and liquid bacterial agent according to claim 5, characterized in that: The method comprises: 1) Strain activation: Pick the strain and streak it onto R2A solid medium, place it in a constant temperature incubator, and culture it at 26°C for 48 hours; 2) Preparation of seed solution: Pick activated bacteria and inoculate them into a large test tube containing seed culture medium, seal it with a rubber stopper, place it on a shaker at 26°C and shake at 150 rpm for 16-24 hours to obtain seed solution; 3) Liquid fermentation: The seed liquid and seed culture medium are then expanded with an inoculation amount of 5-10% by volume, and the culture conditions are 26°C and 150rpm; the culture obtained after 72 hours of culture is the fermentation liquid; the fermentation liquid is centrifuged at 8000rpm for 15 minutes to collect the bacteria.

7. The method according to claim 6, characterized in that The seed culture medium is R2A liquid culture medium, and the formula of the R2A liquid culture medium is: 0.5g yeast extract, 0.5g peptone, 0.5g tyrosine, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.05g potassium dihydrogen phosphate, distilled water is added to make the volume to 1000mL, and sterilized at 121°C for 20min; the formula of the R2A solid culture medium is 15.0g agar added to the R2A liquid culture medium.

8. Use of the Pseudoromonas strain according to claim 1 or the fermentation liquid and liquid inoculum according to claim 5 in synthesizing PHB.

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