Pseudomonas roseonatra strain and application thereof

Abstract

The application discloses a Falsiroseomonas strain and application thereof, the strain is a new species in the Falsiroseomonas genus, and the preservation number is CGMCC No.1.14501.The strain E2-1-a4 obtained by the application is a new species different from known species of the Falsiroseomonas genus, and can produce poly-beta-hydroxybutyrate (PHB). The produced PHB has very wide development and application prospects in medicine, agriculture and packaging materials.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Pseudomonas aeruginosa* and its application in the synthesis of PHB. Background Technology

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

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

[0004] This genus of bacteria possesses diverse metabolic capabilities, utilizing a variety of carbon and nitrogen sources for growth and metabolism. Some strains can utilize simple organic compounds such as glucose and sucrose as carbon sources, and ammonium salts and nitrates as nitrogen sources. When carbon sources are abundant in the environment, but nitrogen, phosphorus, and other nutrients are relatively scarce, Falsiroseomonas will convert excess carbon sources into PHB for storage. In the case of insufficient carbon sources, PHB can be broken down into acetyl-CoA and other substances that enter the tricarboxylic acid cycle, providing energy and a carbon skeleton for the cell, maintaining cellular growth, reproduction, metabolism, and other vital activities. In addition, in high-salt environments or other environments that cause changes in extracellular osmotic pressure, the synthesis and accumulation of PHB by Falsiroseomonas can balance the osmotic pressure inside and outside the cell, preventing cell damage due to osmotic pressure imbalance and ensuring normal cellular physiological functions.

[0005] Poly-β-hydroxybutyrate (PHB) is a linear polymer whose monomer is (R)-3-hydroxybutyric acid, linked by ester bonds. It possesses plastic properties such as thermoplasticity and ductility, allowing for spinning and film fabrication. It is also biodegradable and widely used in various fields. For example, it shows great potential in medical implant materials, serving as a raw material for surgical sutures, patches, and various scaffolds used in surgery. It can also be formulated into gels, microspheres, and nanoparticles for drug delivery. In agriculture, due to its biodegradability, PHB can be used as agricultural mulch to reduce white pollution and as a carrier for fertilizers and pesticides, allowing for slow release. In packaging materials, PHB also exhibits excellent barrier properties, making it suitable for packaging volatile foods such as tea and spices.

[0006] Currently, the main methods for synthesizing PHB are gene synthesis, chemical synthesis, and microbial synthesis. Gene synthesis is relatively complex, while chemical synthesis is costly and generates byproducts that affect PHB quality. In contrast, microbial synthesis can utilize renewable resources as raw materials, such as hydrolysate from plant straw and waste molasses. Furthermore, the synthesis process is mild, requiring no extreme conditions, thus reducing energy consumption and equipment requirements. The synthesized PHB also exhibits good biodegradability, allowing it to be decomposed by microorganisms in the natural environment, reducing environmental pollution. Therefore, the use of microbial synthesis for PHB production has broad application prospects. Summary of the Invention

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

[0008] The *Falsiroseomonas* strain E2-1-a4 provided by this invention was isolated from the soil of Longmu Lake in Ngari Prefecture, Tibet Autonomous Region. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.14501, dated February 19, 2025. Bacterial identification revealed that this strain is a novel species within the genus *Falsiroseomonas*.

[0009] Studies have found that 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, glucamide, D-gluconic acid, L-lactic acid, citric acid, α-ketoglutarate, D-malic acid, L-malic acid, bromosuccinic acid, β-hydroxy-DL-butyric acid, and formic acid as its sole carbon source, exhibiting broad substrate spectrum.

[0010] Furthermore, according to physiological experiments, strain E2-1-a4 can grow under conditions of 15-30℃, pH 6.0-10.0, and NaCl concentration of 0%-2.0%. The optimal growth conditions for strain E2-1-a4 are 26℃, pH 9.0, and NaCl concentration of 0.5%.

[0011] A second objective of this invention is to provide a fermentation broth and liquid inoculum prepared from the aforementioned *Pseudomonas aeruginosa* strain, as well as a method for preparing the same, the method comprising:

[0012] 1) Strain activation: Pick a strain and streak it onto R2A solid medium, place it in a constant temperature incubator, and incubate at 26℃ for 48h.

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

[0014] 3) Liquid fermentation: Then, the seed liquid and seed culture medium are inoculated at a volume ratio of 5-10% for expansion culture. The culture conditions are 26℃ and 150rpm. The culture obtained after 72h is the fermentation broth. The fermentation broth is centrifuged at 8000rpm for 15min and the cells are collected.

[0015] The seed culture medium is R2A liquid culture medium, and the formula of 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, and dilute with distilled water to 1000mL, and sterilize at 121℃ for 20min; the formula of R2A solid culture medium is based on R2A liquid culture medium with the addition of 15.0g agar.

[0016] The third objective of this invention is to provide the application of the above-mentioned *Pseudomonas aeruginosa* strain, as well as the fermentation broth and liquid inoculum prepared from the strain, in the synthesis of PHB.

[0017] Transmission electron microscopy revealed transparent particles in the ultrathin section of strain E2-1-a4, which were identified as PHB.

[0018] Then, following the PHB extraction method, 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 bacterial cells with a fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde, prepare ultrathin sections and observe whether there are transparent particles inside the bacterial cells using transmission electron microscopy; freeze-dry the precipitated bacterial cells and weigh them, and add 30 mL of chloroform and 30 mL of sodium hypochlorite aqueous solution (30%, v / v) per gram of dry cell weight.

[0021] 3) Place the above mixture in a shaker (150 rpm, 30°C) and shake for 90 min.

[0022] 4) The mixture after centrifugation (3000g, 15min) will be separated into three phases: the upper layer is sodium hypochlorite solution, the middle layer is cell debris without PHB, and the lower layer is chloroform with PHB.

[0023] 5) Use a pipette tip to draw up the upper layer of 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 this invention:

[0026] The *Pseudomonas roseum* E2-1-a4 strain of this invention was isolated from the soil on the shore of Longmu Co Salt Lake in Ngari Prefecture, Tibet Autonomous Region. The environment was unpolluted, and it exhibits no ecotoxicity, high safety, and strong stability.

[0027] The *Falsiroseomonas* E2-1-a4 strain of this invention was identified as a new species of the genus *Falsiroseomonas* through 16S rRNA gene sequence analysis, genomic analysis, phylogenetic analysis, and multiple taxonomic analyses.

[0028] The *Pseudomonas roseum* E2-1-a4 strain of this invention is simple to cultivate, has a short cycle, and is highly stable. It can produce PHB, which has a very broad prospect for development and application in medicine, agriculture, and packaging materials. Attached Figure Description

[0029] Figure 1 Morphological image of strain E2-1-a4 under an electron transmission microscope.

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

[0031] Figure 3 Ultrathin section of strain E2-1-a4 under an electron transmission microscope. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0033] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0035] Example 1: Isolation and Identification of Pseudomonas roseum strain E2-1-a4

[0036] a. Soil sample collection: Soil samples were collected from the shore of Longmu Lake in Ngari Prefecture, Tibet Autonomous Region, and brought back to the laboratory at 4°C. The bacterial strains were isolated using the plate dilution coating method.

[0037] b. The culture medium for isolating the strain was R2A solid medium: 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, 15g agar, and distilled water was added to a final volume of 1000mL. The mixture was then sterilized at 121℃ for 20min.

[0038] c. Separation using the plate dilution method: Thoroughly mix the collected soil sample, weigh 10g, and place it in an Erlenmeyer flask containing an appropriate amount of glass beads and 90mL of sterile water. Place the flask on a shaker, shake for 20min, let stand for 5min, and then dilute sequentially using a 10-fold serial dilution method. Take 10g of the diluted sample... -4 10 -5 10 -6 200 μL of each soil dilution was evenly spread onto R2A agar plates. Three plates were spread for each dilution, and the plates were incubated upside down in a 20°C incubator for 14 days. Single colonies with different morphologies were selected for further streaking purification. The purified strains were then transferred to R2A slant agar and stored at 4°C for later use.

[0039] d. Identification of the strain: The purified strain E2-1-a4 was streaked onto an R2A plate, and the colony morphology was observed: the colonies were round, pink, smooth, opaque, and had regular edges. Cell morphology was observed under an electron transmission microscope; the cells were spherical. Figure 1PCR amplicon sequencing was performed using universal primers 27F and 1492R for 16S rRNA, and the resulting sequence is shown in SEQ ID NO.1. Comparison with 16S rRNA sequences of known strains in the Genbank database revealed that strain E2-1-a4 showed the highest similarity (98.60%) to the 16S rRNA gene sequence of *Falsiroseomonas frigidaquae* CW67. Phenotypic and physicochemical characteristics of the strain were analyzed according to Bergey's Manual of Bacteriological Identification and multiple taxonomic methods, and a phylogenetic tree analysis of the 16S rRNA gene sequence was also performed. Figure 2 Genome sequencing analysis confirmed that strain E2-1-a4 is a new species of the genus *Falsiroseomonas* (*Falsiroseomonas* sp. nov.). This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.14501 on February 19, 2025.

[0040] The sequence of SEQ ID NO.1 is as follows:

[0041]

[0042]

[0043] Studies of this strain revealed that 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, glucamide, D-gluconic acid, L-lactic acid, citric acid, α-ketoglutarate, D-malic acid, L-malic acid, bromosuccinic acid, β-hydroxy-DL-butyric acid, and formic acid as its sole carbon source. Furthermore, physiological experiments showed that strain E2-1-a4 can grow at temperatures of 15-30℃, pH values ​​of 6.0-10.0, and NaCl concentrations of 0%-2.0%. The optimal growth conditions for strain E2-1-a4 are 26℃, pH 9.0, and NaCl concentration of 0.5%.

[0044] Example 2: Preparation of fermentation broth for *Pseudomonas roseum* strain E2-1-a4

[0045] a. Strain activation: Select strains and streak them onto R2A solid medium (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, 15g agar, add distilled water to a final volume of 1000mL, pH 7.2-7.4, sterilize at 121℃ for 20min), and incubate at 26℃ for 48h.

[0046] b. Preparation of seed culture: Select activated strains and inoculate them into R2A liquid medium (the formula and preparation are the same as the above R2A solid medium, without adding agar). After culturing at 26℃ and shaking at 150 rpm for 16-24 hours, liquid seeds are obtained.

[0047] c. Liquid fermentation: The culture was expanded by inoculating liquid seed and fermentation medium (formula and preparation are the same as R2A liquid medium above) at a volume ratio of 5-10%. The culture conditions were 26℃, 150 rpm, and shaking culture for 72 h. The resulting culture is the fermentation broth.

[0048] Example 3: Transmission electron microscopy observation of the synthesized PHB strain of *Pseudomonas roseum* E2-1-a4.

[0049] a. Centrifuge the fermentation broth from Example 2 at 8000 rpm for 15 min, discard the supernatant, and collect the precipitated bacterial cells.

[0050] b. Take 20N 30 μL of the precipitated bacterial cells into a 1.5 mL EP tube, fix with 0.2 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8), 2.5% glutaraldehyde, and 1% paraformaldehyde at room temperature for 30 min, then place at 4℃ for 1.5 h (shake the liquid several times during fixation to ensure thorough mixing). Finally, store at 4℃.

[0051] c. Centrifuge the fixative solution from b (6000 rpm, 3 min) and collect the bacterial cells; mix the warm, non-condensing agar with the bacterial cells, cool, remove excess agar, leaving agar clumps containing bacterial cells; then rinse 5 times with 0.1 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8), 5 min each time.

[0052] d. After rinsing, immerse the agar block containing bacterial cells in osmium tetroxide solution (this process should be performed on ice). Observe whether the color of the agar block turns dark brown. After the color changes, observe for another 10 minutes. If the color no longer changes, remove the osmium tetroxide solution. Rinse three times with 0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=8), 5 minutes each time.

[0053] e. Dehydration was performed using ethanol solutions of varying concentrations: 30%, 50%, 70%, 85%, 95%, and 100%. 1 ml of each of these ethanol concentrations was sequentially added to the sample treated with d, and the sample was immersed for 3 minutes. Except for the 100% anhydrous ethanol dehydration step, which was performed at room temperature, all other steps were performed on ice. The 100% anhydrous ethanol dehydration step was repeated 5 times.

[0054] f. Use a mixture of anhydrous ethanol and resin as the penetrant, with a ratio of 1:1 (anhydrous ethanol: resin). Add the prepared penetrant to the dehydrated sample (e), ensuring the sample is completely submerged, and let it stand for 1.5 hours. Afterward, remove the penetrant, add pure resin, and place in a desiccator overnight.

[0055] g. After sample preparation, the samples are ultrathinly sectioned and observed under a transmission electron microscope.

[0056] The observation results showed that in the ultrathin section image of strain E2-1-a4 ( Figure 3 The cells are typically spherical, with numerous spherical transparent granules of varying sizes dispersed within them, consistent with known electron microscopic imaging characteristics of PHB. Because PHB has a low electron density, it appears as transparent regions in the image. Furthermore, the transparent granules in the image are relatively regular spherical in shape and exhibit no clear pattern of distribution within the cell, which also aligns with the characteristic that PHB is stored in granular form within cells.

[0057] To further confirm from a genomic perspective that these transparent particles are indeed PHB, whole-genome sequencing was performed on strain E2-1-a4. Bioinformatics analysis successfully identified key genes related to PHB synthesis: phaA, phbB, and phaC. Specifically, the phaA gene encodes β-ketothiolase, responsible for converting acetyl-CoA to acetoacetyl-CoA; the phbB gene encodes NADPH-dependent acetoacetyl-CoA reductase, which catalyzes the conversion of acetoacetyl-CoA to β-hydroxybutyryl-CoA; and the phaC gene encodes polyhydroxy fatty acid synthase, which catalyzes the polymerization of β-hydroxybutyryl-CoA to form PHB. The presence of these key genes provides strong genetic evidence that strain E2-1-a4 possesses the ability to synthesize PHB, thus further confirming that the transparent particles observed in the ultrathin section are indeed PHB.

[0058] Among them, phaA is located in Scaffold 16 and has a size of 2973bp; phbB is located in Scaffold 9 and has a size of 723bp; phaC is located in Scaffold 16 and has 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 *Pseudomonas roseum* strain E2-1-a4

[0067] The PHB was extracted from the fermentation broth following the PHB extraction procedure. The specific steps are as follows:

[0068] a. Centrifuge the fermentation broth from Example 2 at 8000 rpm for 15 min and collect the precipitated bacterial cells. Freeze-dry the precipitated bacterial cells and weigh them. Add 30 mL of chloroform and 30 mL of sodium hypochlorite aqueous solution (30%, v / v) per gram of dry cell weight.

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

[0070] c. The mixture after centrifugation (3000g, 15min) yields three separate phases: the upper layer is a sodium hypochlorite solution, the middle layer is cell fragments without PHB, and the lower layer is chloroform containing PHB.

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

[0072] e. Finally, ten times the volume of pre-cooled methanol was added to the chloroform phase, and PHB was precipitated.

[0073] Verification has shown that PHB can be extracted from the fermentation broth of *Pseudomonas aeruginosa* strain E2-1-a4 using the above method.

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

Claims

1. A strain of *Pseudomonas roseum*, characterized in that, The strain is *Pseudomonas roseum* (… Falsiroseomonas sp. E2-1-a4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.14501.

2. The fermentation broth and liquid inoculum prepared from the *Pseudomonas aeruginosa* strain according to claim 1.

3. A method for preparing the fermentation broth and liquid inoculant according to claim 2, characterized in that, The method includes: 1) Activation of bacterial strain: Pick a piece of bacterial strain and streak it onto R2A solid medium, place it in a constant temperature incubator, and incubate at 26 ℃ for 48 h; 2) Preparation of seed culture: Pick activated bacterial cells and inoculate them into a large test tube containing seed culture medium, seal the tube with a rubber stopper, and place it on a shaker at 26 ℃ and shake at 150 rpm for 16-24 h to obtain seed culture; 3) Liquid fermentation: The seed liquid and seed culture medium are then inoculated at a volume ratio of 5-10% for large-scale culture at 26℃ and 150 rpm. The culture obtained after 72 h of culture is the fermentation broth. The fermentation broth is centrifuged at 8000 rpm for 15 min and the cells are collected.

4. The method according to claim 3, characterized in that, The seed culture medium is R2A liquid culture medium, and the formula of R2A liquid culture medium is: 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, and distilled water is added to make up to 1000 mL, and sterilized at 121℃ for 20 min; the formula of R2A solid culture medium is based on the R2A liquid culture medium with the addition of 15.0 g agar.

5. The application of the *Pseudomonas pseudorosiformis* strain of claim 1 or the fermentation broth and liquid inoculum of claim 2 in the synthesis of PHB.

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