Shortwave monotypic bacterium strain Bv-xms2024 producing phytorepinephrine and its application

Abstract

This invention belongs to the field of biotechnology, specifically relating to the *Brevundimonas vesicularis* strain Bv-xms2024, which produces phytoene, and its applications. A phytoene-producing *Brevundimonas vesicularis* strain is provided, with the accession number CGMCC No. 30441. This strain can efficiently synthesize phytoene, and neither the bacterial cells nor the fermentation products have significant toxic effects on mice and poultry. It can be used for the industrial production of phytoene and as a feed additive for livestock and poultry, showing promising application prospects in animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a shortwave monocytogenes strain Bv-xms2024 capable of efficiently producing phytoene and its application in the industrial production of phytoene and livestock feed additives. Background Technology

[0002] Phytoene is an important carotenoid with unique antioxidant, anti-inflammatory, and immunomodulatory activities, making it widely used in food, pharmaceuticals, health products, and feed additives. In recent years, with increasing health awareness, market demand for phytoene has continued to grow. However, the limitations of traditional production methods restrict the widespread application of this compound, mainly in the following ways: 1) High production costs: Plant extraction requires large quantities of plant raw materials and complex processes, resulting in high extraction costs; chemical synthesis has strict requirements for reaction conditions and reagent purity, further increasing production costs. 2) Low production efficiency: Plant extraction is limited by growth cycles and environmental conditions, making it unsuitable for large-scale production; chemical synthesis has limited yields and is accompanied by numerous byproducts, affecting product purity. 3) Environmental pollution: Chemical synthesis typically uses organic solvents and catalysts, which may pollute the environment.

[0003] Against this backdrop, microbial fermentation has gradually emerged as a competitive alternative production method. Compared with traditional methods, microbial fermentation has the following significant advantages: 1) High efficiency: Large-scale production can be achieved in a short time. 2) Low cost: The fermentation process is simple and has high resource utilization. 3) Environmentally friendly: It reduces dependence on organic solvents and catalysts, resulting in lower pollution risks.

[0004] Although studies have reported the use of various microbial strains for carotenoid synthesis, strains specifically and efficiently producing phytoene are rare. Furthermore, existing strains still have significant room for improvement in terms of phytoene yield and stability. Therefore, developing a microbial strain capable of efficiently and stably producing phytoene would not only meet market demand but also promote the sustainable development of related industries, possessing significant scientific value and promising application prospects. Summary of the Invention

[0005] The present invention aims to solve the problems of high production cost, low production efficiency and environmental pollution of phytoene in the prior art.

[0006] To address the aforementioned problems, this invention provides a strain of Brevundimonas vesicularis that produces phytoene, named Bv-xms2024, with accession number CGMCC No.30441.

[0007] The present invention also provides a culture of the aforementioned shortwave monoclonal strain Bv-xms2024.

[0008] In some embodiments, the culture is a solid culture of the shortwave monoclonal strain Bv-xms2024.

[0009] In other embodiments, the culture is a liquid culture of the shortwave monoclonal strain Bv-xms2024.

[0010] The present invention also provides a bacterial agent comprising the aforementioned shortwave monoclonal strain Bv-xms2024.

[0011] The bacterial agent can be a solid bacterial agent or a liquid bacterial agent.

[0012] The present invention also provides a feed additive comprising the aforementioned shortwave monoclonal strain Bv-xms2024.

[0013] In some embodiments, the feed additive is the bacterial cells of the shortwave monoclonal strain Bv-xms2024.

[0014] In other embodiments, the feed additive is a culture of the shortwave monoclonal strain Bv-xms2024.

[0015] The feed additive is in solid or liquid form.

[0016] The application of the shortwave monocytogenes strain Bv-xms2024 in the preparation of livestock and poultry feed additives also falls within the scope of this invention.

[0017] The application of the shortwave monocytogenes strain Bv-xms2024 in the production of phytohexene is also within the scope of this invention.

[0018] The present invention also provides a method for producing phytoene, comprising: inoculating the shortwave monoclonal strain Bv-xms2024 into a liquid culture medium and culturing it at 20-30°C and 150-170 rpm for 72-96 hours.

[0019] In the above method, the liquid culture medium can be liquid LB culture medium.

[0020] The *Bacillus shortwave dimorphosus* strain Bv-xms2024 provided by this invention was isolated from contaminants in laboratory LB medium. This strain can grow in LB medium at a suitable temperature range of 15°C to 37°C, with 25°C being the optimal growth temperature. Molecular biological identification showed that its 16S rRNA gene sequence has more than 99% homology with *Bacillus shortwave dimorphosus*. This strain exhibits typical morphological characteristics and specific biochemical properties of *Bacillus shortwave dimorphosus*.

[0021] The shortwave monoclonal strain Bv-xms2024 provided by this invention has the following advantages:

[0022] High production efficiency of phytoene Targeted metabolomics analysis showed that *Bacillus shortwave distillosporus* Bv-xms2024 can efficiently produce phytocyanin while maintaining low byproduct levels. Under optimized fermentation conditions (25℃, 150 rpm, 96 hours), Bv-xms2024 achieved a phytocyanin yield of 477.53 μg / g wet cell volume, significantly higher than other carotenoids such as β-carotene (24.48 μg / g wet cell volume), astaxanthin (11.37 μg / g wet cell volume), and zeaxanthin (10.35 μg / g wet cell volume). Compared to traditional plant extraction and chemical synthesis methods, fermentation of Bv-xms2024 for phytocyanin production can significantly reduce production costs.

[0023] Wide range of industrial applications Shortwave monocytogenes Bv-xms2024 can not only be used in the industrial production of phytoene, but its cells or cultures (such as fermentation broth) can also be directly added as an additive to animals' daily drinking water or feed to supplement phytoene in animals, thereby improving animal health and production performance.

[0024] High animal safety Animal safety evaluation experiments have shown that shortwave monoclonal bacteria Bv-xms2024 showed no toxic reactions in mice and chickens, proving that its application in the food and feed industries is safe and reliable.

[0025] Environmentally friendly Compared with traditional plant extraction and chemical synthesis methods, the fermentation production of phytoene using Bv-xms2024 avoids the use of organic solvents and catalysts, thus reducing the risk of environmental pollution.

[0026] The preservation information of the shortwave monoclonal strain Bv-xms2024 provided by this invention is as follows:

[0027] Biomaterials: Bv-xms2024

[0028] Accession number: CGMCC No. 30441

[0029] Classification and nomenclature: Brevundimonas vesicularis

[0030] Deposit date: April 25, 2024

[0031] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0032] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0033] Figure 1 The colony morphology and Gram staining results of shortwave monocytogenes Bv-xms2024 are shown. A: Colony morphology of Bv-xms2024 after 24 h of growth on LB agar plates; B: Colony morphology of Bv-xms2024 after 96 h of growth on LB agar plates; C: Gram staining results of Bv-xms2024, with a scale bar of 10 micrometers.

[0034] Figure 2 This is an agarose gel electrophoresis image of the 16S rRNA gene amplification product of *B. shortwave monocytogenes* Bv-xms2024; lane M is the DNA marker; the bands in the image are the 16S rRNA gene amplification products, approximately 1400 bp in size.

[0035] Figure 3 Phylogenetic tree of shortwave monocytogenes Bv-xms2024 constructed using 16S rRNA as the standard.

[0036] Figure 4 The growth curve of shortwave monocytogenes Bv-xms2024 was measured in Example 1.

[0037] Figure 5 This shows the relative expression levels of major carotenoid synthesis-related genes (crtE, crtB, crtI, crtY, crtZ, crtW) in the shortwave monocytogenes Bv-xms2024.

[0038] Figure 6 This describes the carotenoid biosynthesis pathway proposed in *Bacillus shortwave diplosporum* Bv-xms2024; where FPP is an abbreviation for Farnesyl pyrophosphate; GGPP is an abbreviation for Geranylgeranylpyrophosphat; and crtE, crtB, crtI, crtY, crtZ, and crtW are genes related to carotenoid synthesis.

[0039] Figure 7 The chromatogram shows phytoene in the extract of *Bacillus shortwave diplosporum* Bv-xms2024.

[0040] Figure 8 The chromatogram shows β-carotene in the extract of *Bacillus shortwave diplosporum* Bv-xms2024.

[0041] Figure 9The chromatogram shows astaxanthin in the extract of *Bacillus shortwave diplosporum* Bv-xms2024.

[0042] Figure 10 Chromatoxanthrin in the extract of *Bacillus shortwave diplosporum* Bv-xms2024 is shown in the image.

[0043] Sequence Description

[0044] SEQ ID NO:1 is the nucleotide sequence of the 16S rRNA gene of *Bacillus shortwave diplosporum* Bv-xms2024;

[0045] SEQ ID NO:2 is the nucleotide sequence of primer 27F;

[0046] SEQ ID NO:3 is the nucleotide sequence of primer 1492R;

[0047] SEQ ID NO:4 is the nucleotide sequence of primer CrtE-F;

[0048] SEQ ID NO:5 is the nucleotide sequence of primer CrtE-R;

[0049] SEQ ID NO:6 is the nucleotide sequence of primer CrtB-F;

[0050] SEQ ID NO:7 is the nucleotide sequence of primer CrtB-R;

[0051] SEQ ID NO:8 is the nucleotide sequence of primer CrtI-F;

[0052] SEQ ID NO:9 is the nucleotide sequence of primer CrtI-R;

[0053] SEQ ID NO:10 is the nucleotide sequence of primer CrtY-F;

[0054] SEQ ID NO:11 is the nucleotide sequence of primer CrtY-R;

[0055] SEQ ID NO:12 is the nucleotide sequence of primer CrtZ-F;

[0056] SEQ ID NO:13 is the nucleotide sequence of primer CrtZ-R;

[0057] SEQ ID NO:14 is the nucleotide sequence of primer CrtW-F;

[0058] SEQ ID NO:15 is the nucleotide sequence of primer CrtW-R;

[0059] SEQ ID NO:16 is the nucleotide sequence of primer 16S-F;

[0060] SEQ ID NO:17 is the nucleotide sequence of primer 16S-R. Detailed Implementation

[0061] The following implementation plan is provided:

[0062] 1. A strain of Brevundimonas vesicularis that produces phytoene, with accession number CGMCC No. 30441.

[0063] 2. Cultures of the shortwave monoclonal bacteria strain described in Implementation Scheme 1.

[0064] 3. The culture described in Implementation Scheme 2 is a solid culture of the aforementioned shortwave monoclonal strain.

[0065] 4. The culture described in Implementation Scheme 2 is a liquid culture of the aforementioned shortwave monoclonal strain.

[0066] 5. A microbial agent comprising the shortwave monocytogenes strain described in embodiment 1.

[0067] 6. The microbial agent described in Implementation Scheme 5 is a solid microbial agent or a liquid microbial agent.

[0068] 7. A feed additive comprising the shortwave monocytogenes strain described in embodiment 1.

[0069] 8. The feed additive described in Implementation Scheme 7 is the bacterial cell of the aforementioned shortwave monoclonal strain.

[0070] 9. The feed additive described in Implementation Scheme 7 is a culture obtained by culturing the shortwave monoclonal strain in a solid culture medium.

[0071] 10. The feed additive described in Implementation Scheme 7 is a fermentation broth obtained by culturing the shortwave monocytogenes strain in a liquid culture medium.

[0072] 11. The feed additive described in Implementation Scheme 7 is in solid or liquid form.

[0073] 12. Application of the shortwave monoclonal strain described in Implementation Scheme 1 in the preparation of livestock and poultry feed additives.

[0074] 13. Application of the shortwave monoclonal strain described in Implementation Scheme 1 in the production of phytohexene.

[0075] 14. A method for producing phytoene, comprising: inoculating the *Syntrophus shortwave monocytogenes* strain described in embodiment 1 into a liquid culture medium and culturing it at 20–30°C and 150–170 rpm for 72–96 hours.

[0076] 15. The method of embodiment 14, wherein the liquid culture medium is liquid LB culture medium.

[0077] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way.

[0078] Unless otherwise specified, all reagents used in the following examples are conventional reagents in the art, commercially available, or prepared according to conventional methods in the art. All experimental methods not specifically described in the following examples are conventional experimental methods in the art, and can be found in relevant experimental manuals, publicly available literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0079] Example 1. Isolation and identification of shortwave monoclonal bacteria Bv-xms2024

[0080] 1. Isolation and purification of bacterial strains

[0081] Shortwave monoclonal bacteria Bv-xms2024 were isolated from contaminants in laboratory LB agar medium in 2024. Contaminant samples were collected using sterile swabs and inoculated onto LB agar plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride).

[0082] The culture was prepared on 1.5% agar (pH 7.0) and incubated at 25°C for 48 hours. A pure culture strain was obtained through continuous single-colony isolation and purification, and named Bv-xms2024.

[0083] 2. Morphological observation of the strain

[0084] The purified Bv-xms2024 strain was inoculated onto LB agar plates and incubated at 25°C for 24 hours and 96 hours, after which colony morphology was observed. Changes in colony morphology are shown below. Figure 1 As shown. After 24 hours of incubation, the colonies are round, with smooth edges, a raised center, and a color ranging from transparent to slightly white. Figure 1 A). After 96 hours of incubation, the colonies gradually turned orange-yellow and became slightly viscous. Figure 1 B). Gram staining results showed that strain Bv-xms2024 was a short rod-shaped Gram-negative bacterium. Figure 1 C).

[0085] 3. Molecular biological identification

[0086] Bv-xms2024 strain was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 72 hours. The bacterial culture was then centrifuged at 12000 rpm for 10 min to collect the bacterial cells. Total bacterial DNA was extracted using a bacterial DNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D3146). Using the extracted total DNA as a template, the 16S rRNA gene of Bv-xms2024 strain was amplified by polymerase chain reaction (PCR) using primers 27F / 1492R. The nucleotide sequences of the primers are as follows:

[0087] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:2)

[0088] Reverse primer 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO:3)

[0089] PCR reaction mixture (50 μL): 2×Taq PCR Mix, 25 μL; forward primer 27F (10 μM), 2 μL; reverse primer 1492R (10 μM), 2 μL; template DNA, 1 μL; ddH2O, 20 μL. The negative control was performed using ddH2O instead of template DNA.

[0090] PCR reaction program: 94℃ pre-denaturation for 5 min; (94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min) 30 cycles; 72℃ final extension for 10 min.

[0091] After the reaction, the amplification products were detected by 1% agarose gel electrophoresis. Figure 2 As shown, the amplified product band size is approximately 1400 bp, which is in line with expectations.

[0092] The amplified products were sequenced, and the determined sequences were compared with the NCBI GenBank database using BLAST. The results showed that the 16S rRNA gene sequence of strain Bv-xms2024 had 99.5% similarity to that of *B. shortwave*. A phylogenetic tree was constructed using MEGA11 software with a neighbor-joining method. The results showed that strain Bv-xms2024 clustered with other strains of the *B. shortwave* genus. Figure 3 Therefore, strain Bv-xms2024 was confirmed to be Brevundimonas vesicularis.

[0093] The nucleotide sequence (1332 bp) of the 16S rRNA gene of strain Bv-xms2024:

[0094] GTGGTCGCCTGCCTCCTTGCGGTCAGCGCAGCGCCTTCGGGTAGAACCAA

[0095] CTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACC

[0096] GCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGCCCTCGAG

[0097] TTGCAGAGGACAATCCGAACTGAGACGACTTTTAAGGATTAACCCTCTGT

[0098] AGTCGCCATTGTAGCACGTGTGTAGCCCACCCTGTAAGGGCCATGAGGAC

[0099] TTGACGTCATCCCCACCTTCCTCCGGCTTAGCACCGGCAGTCCCATTAGA

[0100] GTTCCCAACTAAATGATGGCAACTAATGGCGAGGGTTGCGCTCGTTGCGG

[0101] GACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACC

[0102] TGTGTCCTAGTCCCCGAAGGGAAAGCCAGATCTCTCTGGCGGTCCAGGCA

[0103] TGTCAAAAGGTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGC

[0104] TCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCG

[0105] ACCGTACTCCCCAGGCGGATTGCTTAATGCGTTAGCTGCGTCACCGAAAT

[0106] GCATGCATCCCGACAACTAGCAATCATCGTTTACGGCGTGGACTACCAGG

[0107] GTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTAAT

[0108] GAGCCAGTGTGTCGCCTTCGCCACTGGTGTTCTTCCGAATATCTACGAAT

[0109] TTCACCTCTACACTCGGAGTTCCACACACCTCTCTCATACTCAAGACACC

[0110] CAGTATCAAAGGCAATTCCGAGGTTGAGCCCCGGGATTTCACCCCTGACT

[0111] TAAATGTCCGCCTACGCTCCCTTTACGCCCAGTAATTCCGAGCAACGCTA

[0112] GCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCT

[0113] TCTCCGGGTACCGTCATTATCGTCCCCGGTGAAAGAATTTTACAATCCTA

[0114] AGACCTTCATCATTCACGCGGCATGGCTGCGTCAGGCTTTCGCCCATTGC

[0115] GCAAGATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGT

[0116] CCCAATGTGGCTGATCATCCTCTCAGACCAGCTACTGATCGTCGCCTTGG

[0117] TGAGCCTTTACCTCACCAACTAGCTAATCAGACGCGGGCCGCTCTAAAGG

[0118] CGATAAATCTTTCCCCCGAAGGGCACATTCGGTATTAGCACAAGTTTCCC

[0119] TGAGTTATTCCGAACCTAAAGGCACGTTCCCACGTGTTACTCACCCGTCC

[0120] GCCACTAACTCCGAAGAGTTCGTTCGACTTGC(SEQ ID NO:1)

[0121] 4. Determination of physiological and biochemical characteristics

[0122] The physiological and biochemical characteristics of strain Bv-xms2024 were detected. The results of the catalytic enzyme test were positive, the oxidase test was negative, the glucose and sucrose fermentation test was positive, the lactose fermentation test was negative, and the nitrate reduction test was positive.

[0123] 5. Determination of bacterial growth curves

[0124] The Bv-xms2024 strain was inoculated into liquid LB medium and cultured at 25°C and 170 rpm. Its growth curve was then measured. The results are as follows: Figure 4 As shown, the hysteresis phase lasts 0-40 hours; the logarithmic growth phase lasts 40-60 hours, during which the bacterial solution gradually turns orange-red; the stationary phase lasts 60-64 hours, during which the number of viable bacteria remains stable; and the decline phase lasts 64 hours, during which the number of viable bacteria gradually decreases.

[0125] 6. Preservation of microbial strains

[0126] The strain Bv-xms2024 was deposited at the China General Microbiological Culture Collection Center (CGMCC) under the name Bv-xms2024, accession number CGMCC No. 30441, and deposit date of April 25, 2024.

[0127] Example 2. Production of phytoene using shortwave monocytogenes Bv-xms2024

[0128] 1. Optimization of fermentation culture conditions

[0129] The LB medium formula is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0. The medium is then autoclaved at 121°C for 20 minutes before use.

[0130] Shortwave monocytogenes Bv-xms2024 was revived from storage and inoculated onto LB agar plates, then cultured at 25°C for 48 hours. Single colonies were picked and inoculated into 50 mL of liquid LB medium, and cultured with shaking at 25°C and 170 rpm for 24 hours to prepare a seed culture. The seed culture was inoculated into 400 mL of LB medium at a rate of 2% (v / v), and cultured at temperatures of 15°C, 20°C, 25°C, 30°C, and 37°C; at shaking speeds of 100 rpm, 150 rpm, and 170 rpm; and for incubation times of 48 hours, 72 hours, and 96 hours. Viable cell counts showed that the optimal fermentation conditions for strain Bv-xms2024 were 25°C, 170 rpm, and 96 hours.

[0131] 2. Extraction and quantitative analysis of phytoene

[0132] Shortwave monocytogenes Bv-xms2024 was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 96 hours. After incubation, 400 mL of culture was centrifuged at 5000 rpm for 10 minutes, and the bacterial pellet was collected and weighed. 10 mL of acetone was added to each gram of wet bacterial cells, and the mixture was then extracted overnight in the dark at 25°C and 170 rpm with shaking until the bacterial color lightened. The extract was filtered through Whatman filter paper, and the filtrate was collected for quantitative analysis.

[0133] Sixty-eight carotenoid metabolites in the filtrate were quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS / MS).

[0134] Liquid phase conditions mainly include:

[0135] Column: YMC C30 (3μm, 100mm x 2.0mm id);

[0136] Mobile phases: Phase A, methanol / acetonitrile (1:3, v / v) with 0.01% BHT and 0.1% formic acid; Phase B, methyl tert-butyl ether with 0.01% BHT.

[0137] Gradient elution program: A / B, 0 min 100:0 (V / V), 3 min 100:0 (V / V), 5 min 30:70 (V / V), 9 min 5:95 (V / V), 10 min 100:0 (V / V), 11 min 100:0 (V / V);

[0138] Flow rate: 0.8 mL / min; column temperature: 28 °C; injection volume: 2 μL.

[0139] Mass spectrometry conditions mainly include:

[0140] The atmospheric pressure chemical ionization source (APCI) operates at a temperature of 350°C with a curtain gas (CUR) pressure of 25 psi. In the Q-Trap 6500+, each ion pair is scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0141] Figures 7-10 The liquid chromatograms of phytoene, β-carotene, astaxanthin, and zeaxanthin in the extract of *Bacillus shortwave dispiritus* Bv-xms2024 are shown. The results indicate that phytoene had the highest content, reaching 477.53 μg / g wet cell volume; other carotenoids, such as β-carotene (24.48 μg / g wet cell volume), astaxanthin (11.37 μg / g wet cell volume), and zeaxanthin (10.35 μg / g wet cell volume), had relatively lower contents.

[0142] 3. Expression analysis of genes related to carotenoid synthesis

[0143] The temporal expression of carotenoid synthesis-related genes (crtE, crtB, crtI, crtY, crtZ, and crtW) in *Bacillus shortwave dispirin* Bv-xms2024 was analyzed using real-time quantitative PCR (RT-qPCR) to reveal the molecular mechanism of high phytorepinephrine production. The PCR primers for each related gene are shown in Table 1.

[0144] Table 1

[0145]

[0146]

[0147] Shortwave monocytogenes Bv-xms2024 was inoculated into liquid LB medium and cultured at 25°C and 170 rpm. Bacterial cells were collected after 24, 48, 72, and 96 hours of culture. Total RNA was extracted from the cells using TRIzol reagent and reverse transcribed into cDNA. Using cDNA as a template, quantitative real-time PCR was performed on the primer pairs CrtE-F / R, CrtB-F / R, CrtI-F / R, CrtY-F / R, CrtZ-F / R, and CrtW-F / R, as shown in Table 1, to analyze the expression levels of carotenoid synthesis-related genes (crtE, crtB, crtI, crtY, crtZ, and crtW) at different culture times. The 16S rRNA gene (GenBank accession number: NR_037104) was used as an internal reference gene, and its qPCR primers are as follows:

[0148] 16s-F:AGAGTTTGATCMTGGCTCAG(SEQ ID NO:16)

[0149] 16s-R: CGGTTACCTTGTTACGACTT(SEQ ID NO:17)

[0150] PCR reaction system (50 μL): 2×Hieff Universal Blue qPCR Master Mix 25 μL; ddH2O 20 μL; corresponding upstream primer (10 μM) 2 μL; corresponding downstream primer (10 μM) 2 μL; and template DNA 1 μL. PCR reaction program: 50℃ UDG activation for 2 min; 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s; 60℃ annealing / extension for 30 s; 40 cycles, with fluorescence signal collected after each cycle.

[0151] After the reaction was completed, the Ct (Cycles threshold) value was obtained. The data was then analyzed and processed using a 2... -△△Ct The calculation formula calculates the relative expression level of each gene.

[0152] RT-qPCR analysis results are as follows Figure 5 As shown: After 48 hours of culture, the expression of crtE and crtB genes was significantly upregulated, promoting the efficient synthesis of phytopenic acid; after 96 hours of culture, the expression of crtI, crtY, crtW, and crtZ genes gradually increased, and were associated with the synthesis of other carotenoids (such as astaxanthin). The carotenoid synthesis pathway is as follows: Figure 6 As shown.

[0153] Example 3. Animal safety evaluation of shortwave monoclonal bacteria Bv-xms2024

[0154] 1. Safety evaluation in mice

[0155] Shortwave monocytogenes Bv-xms2024 was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 96 hours. The culture was centrifuged at 5000 rpm for 10 minutes to collect the bacterial cells. The bacterial cells were resuspended in sterile physiological saline to prepare concentrations of 1 × 10⁻⁶. 9 CFU / mL, 1 × 10 10 CFU / mL and 1 × 10 11 Bv-xms2024 bacterial suspension at CFU / mL.

[0156] Forty healthy Kunming mice were randomly divided into four groups of ten each, with half males and half females. The healthy Kunming mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The four groups of mice were treated as follows:

[0157] Control group: 0.2 mL of sterile saline was administered by gavage daily;

[0158] Low-dose group (LG group): 0.2 mL orally per day, 1 × 10 9 Bv-xms2024 bacterial suspension at CFU / mL;

[0159] Medium-dose group (MG group): 0.2 mL 1 × 10⁻⁶ mg orally daily. 10 Bv-xms2024 bacterial suspension at CFU / mL;

[0160] High-dose group (HG group): 0.2 mL orally per day, 1 × 10 11 Bv-xms2024 bacterial suspension at CFU / mL.

[0161] The experiment lasted for 14 days, and the mice were weighed and their health status was recorded every 3 days.

[0162] Results: Throughout the experiment, all mice remained healthy with no significant abnormal behaviors (such as ruffled hair or decreased appetite); as shown in Table 2, there was no significant difference in weight gain trends between the control group and each dosage group (P>0.05). Therefore, oral administration of the Bv-xms2024 strain had no toxic effects on mice.

[0163] Table 2

[0164]

[0165] Note: The values ​​in the table represent the mean ± standard deviation.

[0166] 2. Chicken safety evaluation

[0167] Shortwave monoclonal bacteria Bv-xms2024 were inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 96 hours to obtain a Bv-xms2024 bacterial suspension.

[0168] Twenty 42-day-old SPF chickens were randomly divided into a control group and an experimental group, with 10 chickens in each group. The SPF chickens were purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd. in Beijing. The chickens in the control group and the experimental group were treated as follows:

[0169] Control group: drank normal drinking water daily;

[0170] Experimental group: Drink a suspension of shortwave monocytogenes Bv-xms2024 cultured in LB for 96 hours daily as a substitute for drinking water.

[0171] Chickens underwent a one-week acclimatization period before the experiment. The experiment lasted for 14 days, with weight and health status recorded every 3 days.

[0172] result:

[0173] Throughout the experiment, no health problems, deaths, or illnesses were observed in any of the chickens. As shown in Table 3, the weight gain trends of the control group and the experimental group were similar, with no significant difference (P>0.05). The flock maintained normal feed intake and activity, indicating that the administration of Bv-xms2024 via drinking water had no significant toxic effects on poultry.

[0174] Table 3

[0175]

[0176] Note: The values ​​in the table represent the mean ± standard deviation.

[0177] The above experiments show that shortwave monocytogenes Bv-xms2024 and its fermentation products, as feed additives, have no adverse effects on the health of mice and poultry and have good safety.

Claims

1. A strain of Brevundimonas vesicularis that produces phytoene, with accession number CGMCC No. 30441.

2. The culture of the Brevundimonas vesicularis strain as described in claim 1.

3. A bacterial agent comprising the *Brevundimonas vesicularis* strain as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent can be a solid bacterial agent or a liquid bacterial agent.

5. A feed additive comprising the Brevundimonas vesicularis strain as described in claim 1.

6. The feed additive according to claim 5, characterized in that, The feed additive is the bacterial cells or culture of the *Brevundimonas vesicularis* strain.

7. The feed additive according to claim 5, characterized in that, The feed additive is in solid or liquid form.

8. The use of the Brevundimonas vesicularis strain as described in claim 1 in the preparation of livestock and poultry feed additives.

9. The use of the Brevundimonas vesicularis strain according to claim 1 in the production of phytohexene.

10. A method for producing phytoene, comprising: The *Brevundimonas vesicularis* strain described in claim 1 was inoculated into liquid culture medium and cultured at 20–30°C and 150–170 rpm for 72–96 hours.

Images