Brevundimonas strain Bv-xms2024 for producing phytoene and application of brevundimonas strain Bv-xms2024

Abstract

The invention belongs to the technical field of biology, and particularly relates to a brevundimonas strain Bv-xms2024 for producing phytoene and application of the brevundimonas strain Bv-xms2024. The invention provides a Brevundimonas veicularis strain for producing phytoene, and the preservation number of the Brevundimonas veicularis strain is CGMCC (China General Microbiological Culture Collection Center) No.30441. The invention further provides a preparation method of the Brevundimonas veicularis strain for producing phytoene. The strain can efficiently synthesize phytoene, and thalli and fermentation products have no obvious toxic effect on mice and poultry, can be used for industrial production of phytoene and livestock and poultry feed additives, and have good application prospects in the field of animal breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Brevundimonas strain Bv-xms2024 capable of efficiently producing phytoene and its applications in industrial production of phytoene and livestock and poultry feed additives. Background Art

[0002] Phytoene is an important carotenoid. Its unique biological activities such as antioxidant, anti-inflammatory and immunomodulatory effects have led to its wide application in fields such as food, medicine, health products and feed additives. In recent years, with the improvement of health consumption awareness, the market demand for phytoene has been continuously increasing. However, the limitations of traditional production methods have restricted the widespread application of this compound, mainly manifested in the following aspects: 1) High production cost: Plant extraction requires a large amount 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 the growth cycle and environmental conditions and cannot meet the needs of large-scale production; the yield of chemical synthesis is limited, and a large number of by-products are generated, affecting the purity of the product. 3) Environmental pollution problems: Organic solvents and catalysts are usually used in the chemical synthesis process, and these substances may cause pollution to the environment.

[0003] Under this background, microbial fermentation has gradually become 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 the resource utilization rate is high. 3) Environmental protection: Reducing the dependence on organic solvents and catalysts, with a lower pollution risk.

[0004] Although many microbial strains have been reported in the literature for the synthesis of carotenoids, strains specifically for efficiently producing phytoene are very rare. In addition, there is still much room for improvement in the yield and stability of phytoene in existing strains. Therefore, developing a microbial strain capable of efficiently and stably producing phytoene can not only meet the market demand but also promote the sustainable development of related industries, which has important scientific value and good 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 solve the above problems, the present invention provides a Brevundimonas vesicularis strain producing phytoene, named Bv-xms2024, and its preservation number is CGMCC No. 30441.

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

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

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

[0010] The present invention also provides a microbial agent, which comprises the Brevundimonas strain Bv-xms2024.

[0011] The microbial agent is a solid microbial agent or a liquid microbial agent.

[0012] The present invention also provides a feed additive, which comprises the Brevundimonas strain Bv-xms2024.

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

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

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

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

[0017] The application of the Brevundimonas strain Bv-xms2024 in the production of phytoene also falls within the scope of the present invention.

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

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

[0020] The Brevundimonas strain Bv-xms2024 provided by the present invention was isolated from the contaminants of a laboratory LB medium. This strain can grow in the LB medium, and the suitable temperature range is 15 °C to 37 °C, with 25 °C being the optimal growth temperature. Molecular biological identification shows that the homology of its 16S rRNA gene sequence with the genus Brevundimonas is higher than 99%. This strain exhibits typical morphological characteristics and specific biochemical characteristics of Brevundimonas.

[0021] The Brevundimonas strain Bv-xms2024 provided by the present invention has the following advantages:

[0022] High production efficiency of lycopene : Targeted metabolomics analysis shows that Brevundimonas Bv-xms2024 can maintain a low by-product level while efficiently producing phytoene. When Bv-xms2024 is cultured under optimized fermentation conditions (25 °C, 150 rpm, 96 hours), the yield of phytoene is as high as 477.53 μg / g wet cell mass, far higher than the contents of other carotenoids such as β-carotene (24.48 μg / g wet cell mass), astaxanthin (11.37 μg / g wet cell mass), and zeaxanthin (10.35 μg / g wet cell mass). Compared with traditional plant extraction methods and chemical synthesis methods, using Bv-xms2024 to ferment and produce phytoene can greatly reduce production costs.

[0023] Widely used in industry : Brevundimonas Bv-xms2024 can not only be used for the industrial production of phytoene, but its cells or cultures (such as fermentation broth) can also be directly added as additives to the daily drinking water or feed of animals to supplement phytoene in the animal body, thereby improving the health level and production performance of animals.

[0024] High animal safety : Animal safety evaluation experiments have proved that no toxic reactions were observed in mice and chickens with Brevundimonas Bv-xms2024, demonstrating its safe and reliable application in the fields of food and feed.

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

[0026] The preservation information of the Brevundimonas strain Bv-xms2024 provided by the present invention is as follows:

[0027] Biological material: Bv-xms2024

[0028] Preservation number: CGMCC No. 30441

[0029] Taxonomic name: Brevundimonas vesicularis

[0030] Preservation date: April 25, 2024

[0031] Preservation unit: China General Microbiological Culture Collection Center

[0032] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, China Description of the Drawings

[0033] Figure 1 Showing the colony morphology and Gram staining results of Brevundimonas sp. Bv-xms2024; A: Colony morphology of Bv-xms2024 grown on LB agar plate for 24 h; B: Colony morphology of Bv-xms2024 grown on LB agar plate for 96 h; C: Gram staining result of Bv-xms2024, the scale bar is 10 μm

[0034] Figure 2 Agarose gel electrophoresis pattern of the 16S rRNA gene amplification product of Brevundimonas sp. Bv-xms2024; Lane M is DNA Marker; the band in the figure is the 16S rRNA gene amplification product, with a size of about 1400 bp

[0035] Figure 3 Phylogenetic tree of Brevundimonas sp. Bv-xms2024 constructed using 16S rRNA as a standard

[0036] Figure 4 Growth curve of Brevundimonas sp. Bv-xms2024 measured in Example 1

[0037] Figure 5 Showing the relative expression levels of the main carotenoid biosynthesis-related genes (crtE, crtB, crtI, crtY, crtZ, crtW) in Brevundimonas sp. Bv-xms2024

[0038] Figure 6 Carotenoid biosynthesis pathway proposed in Brevundimonas sp. Bv-xms2024; where FPP is the abbreviation of Farnesyl pyrophosphate, representing farnesyl pyrophosphate; GGPP is the abbreviation of Geranylgeranylpyrophosphat, representing geranylgeranyl pyrophosphate; crtE, crtB, crtI, crtY, crtZ, crtW are carotenoid biosynthesis-related genes

[0039] Figure 7 Chromatogram of phytoene in the extract of Brevundimonas sp. Bv-xms2024

[0040] Figure 8 Chromatogram of β-carotene in the extract of Brevundimonas sp. Bv-xms2024

[0041] Figure 9Chromatogram of astaxanthin in the extract of Brevundimonas sp. Bv-xms2024

[0042] Figure 10 Chromatogram of zeaxanthin in the extract of Brevundimonas sp. Bv-xms2024

[0043] Sequence Listing

[0044] SEQ ID NO:1 is the nucleotide sequence of the 16S rRNA gene of Brevundimonas sp. 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 manners

[0061] The following implementation manners are provided:

[0062] 1. A Brevundimonas vesicularis strain producing lycopene, with a deposit number of CGMCC No. 30441.

[0063] 2. A culture of the Brevundimonas vesicularis strain described in implementation manner 1.

[0064] 3. The culture described in implementation manner 2 is a solid culture of the Brevundimonas vesicularis strain.

[0065] 4. The culture described in implementation manner 2 is a liquid culture of the Brevundimonas vesicularis strain.

[0066] 5. A bacterial agent comprising the Brevundimonas vesicularis strain described in implementation manner 1.

[0067] 6. The bacterial agent described in implementation manner 5 is a solid bacterial agent or a liquid bacterial agent.

[0068] 7. A feed additive comprising the Brevundimonas vesicularis strain described in implementation manner 1.

[0069] 8. The feed additive described in implementation manner 7 is the cells of the Brevundimonas vesicularis strain.

[0070] 9. The feed additive described in implementation manner 7 is a culture obtained by culturing the Brevundimonas vesicularis strain in a solid medium.

[0071] 10. The feed additive described in implementation manner 7 is a fermentation broth obtained by culturing the Brevundimonas vesicularis strain in a liquid medium.

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

[0073] 12. Use of the Brevundimonas vesicularis strain described in implementation manner 1 in the preparation of livestock and poultry feed additives.

[0074] 13. Use of the Brevundimonas vesicularis strain described in implementation manner 1 in the production of lycopene.

[0075] 14. A method for producing lycopene, which includes: inoculating the Brevundimonas vesicularis strain described in implementation manner 1 into a liquid medium and culturing at 20 - 30 °C and 150 - 170 rpm for 72 - 96 hours.

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

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

[0078] Reagents not specifically described in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art. Experimental methods not specifically described in the following examples are all conventional experimental methods in the art, and relevant experimental manuals, well-known literatures or manufacturer's instructions can be referred to. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0079] Example 1. Isolation and identification of Brevundimonas sp. Bv-xms2024

[0080] 1. Isolation and purification of the strain

[0081] Brevundimonas sp. Bv-xms2024 was isolated from laboratory LB medium contaminants in 2024. The contaminant sample was collected with a sterile cotton swab and inoculated on an LB agar plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride,

[0082] 1.5% agar, pH 7.0), and cultured at 25 °C for 48 hours. Through continuous isolation and purification of single colonies, a pure culture strain was obtained and named Bv-xms2024.

[0083] 2. Morphological observation of the strain

[0084] The purified Brevundimonas sp. Bv-xms2024 strain was inoculated on an LB agar plate, and the colony morphology was observed after culturing at 25 °C for 24 hours and 96 hours, respectively. The changes in colony morphology are as Figure 1 shown. After culturing for 24 hours, the colonies were round, with smooth edges, raised centers, and the color was transparent to slightly white ( Figure 1 A). After culturing for 96 hours, the colony color gradually changed to orange-yellow, and the texture was slightly viscous ( Figure 1 B). The results of Gram staining showed that the Brevundimonas sp. Bv-xms2024 strain was a short rod-shaped Gram-negative bacterium ( Figure 1 C).

[0085] 3. Molecular biological identification

[0086] The Bv-xms2024 strain was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 72 hours. After that, the bacterial solution was centrifuged at 12,000 r / min for 10 min to collect the bacterial cells. The total DNA of the bacteria was extracted using a bacterial DNA extraction kit (product of Guangzhou Meiji Biotechnology Co., Ltd., catalog number D3146). Using the extracted total DNA as a template, the 16S rRNA gene of the 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 system (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. For the negative control, ddH2O was used instead of template DNA.

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

[0091] After the reaction, the amplification products were detected by 1% agarose gel electrophoresis. As Figure 2 shown, the band size of the amplification products was approximately 1400 bp, which was consistent with the expectation.

[0092] The amplification products were sequenced, and the determined sequences were compared with the NCBI GenBank database by BLAST. The results showed that the similarity of the 16S rRNA gene sequence of the Bv-xms2024 strain to the genus Brevundimonas was 99.5%. The MEGA11 software was used to construct a phylogenetic tree by the Neighbor-Joining method. The results showed that the Bv-xms2024 strain clustered with other strains of the genus Brevundimonas ( Figure 3 ), so it was confirmed that the Bv-xms2024 strain was Brevundimonas vesicularis.

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

[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 catalase test were positive, the results of the oxidase test were negative, the results of the glucose and sucrose fermentation tests were positive, the results of the lactose fermentation test were negative, and the results of the nitrate reduction test were positive.

[0123] 5. Determination of the growth curve of the strain

[0124] Strain Bv-xms2024 was inoculated into liquid LB medium and cultured at 25 °C and 170 rpm, and its growth curve was determined. The results are as Figure 4 shown. Lag phase: 0 - 40 hours; Logarithmic growth phase: 40 - 60 hours, and the bacterial liquid gradually turned orange-red; Stationary phase: 60 - 64 hours, and the number of viable bacteria was stable; Decline phase: after 64 hours, the number of viable bacteria gradually decreased.

[0125] 6. Preservation of the strain

[0126] Strain Bv-xms2024 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation name was Bv-xms2024, the preservation number was CGMCC No. 30441, and the preservation date was April 25, 2024.

[0127] Example 2. Production of lycopene by Brevundimonas sp. Bv-xms2024

[0128] 1. Optimization of fermentation culture conditions

[0129] Formulation of LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0. The medium was autoclaved at 121 °C for 20 min and then used.

[0130] After resuscitating Brevundimonas sp. Bv-xms2024 from preservation, it was inoculated onto an LB agar plate and cultured at 25°C for 48 hours. A single colony was 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 solution. The seed solution was inoculated into 400 mL of LB medium at an inoculation amount of 2% (v / v). The culture temperatures were set at 15°C, 20°C, 25°C, 30°C, and 37°C; the rotation speeds were 100 rpm, 150 rpm, and 170 rpm; and the culture times were 48 hours, 72 hours, and 96 hours. The viable cell count of the bacterial solution was performed, and the results showed that the optimal fermentation conditions for the Bv-xms2024 strain were: 25°C, 170 rpm, and 96 hours.

[0131] 2. Extraction and Quantitative Analysis of Lycopene

[0132] Brevundimonas sp. Bv-xms2024 was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 96 hours. After the culture ended, 400 mL of the culture solution was centrifuged at 5000 rpm for 10 minutes to collect the bacterial cell precipitate, and the wet weight of the bacterial cells was weighed and recorded. 10 mL of acetone was added to each gram of wet bacterial cells, and then it was shaken and extracted overnight under dark conditions at 25°C and 170 rpm until the color of the bacterial cells became lighter. The extract was filtered through Whatman filter paper, and the filtrate was collected for quantitative analysis.

[0133] A liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantitatively analyze 68 carotenoid metabolites in the filtrate.

[0134] The liquid phase conditions mainly included:

[0135] Chromatographic column: YMC C30 (3 μm, 100 mm x 2.0 mm i.d.);

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

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

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

[0139] The mass spectrometry conditions mainly included:

[0140] The temperature of the Atmospheric Pressure Chemical Ionization Source (APCI) is 350 °C, and the Curtain Gas (CUR) is 25 psi. In the Q-Trap 6500+, each ion pair is scanned and detected according to the 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 Brevundimonas sp. Bv-xms2024 are shown. The results indicate that the content of phytoene is the highest, reaching 477.53 μg / g wet cells; the contents of other carotenoids such as β-carotene (24.48 μg / g wet cells), astaxanthin (11.37 μg / g wet cells), and zeaxanthin (10.35 μg / g wet cells) are relatively low.

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

[0143] The temporal expression analysis of genes related to carotenoid synthesis (crtE, crtB, crtI, crtY, crtZ, crtW) in Brevundimonas sp. Bv-xms2024 was carried out by real-time quantitative PCR (RT-qPCR) to reveal the molecular mechanism of high phytoene production. The PCR primers for each related gene are shown in Table 1.

[0144] Table 1

[0145]

[0146]

[0147] Brevundimonas sp. Bv-xms2024 was inoculated into liquid LB medium and cultured at 25 °C and 170 rpm. The cells were collected after culturing for 24 hours, 48 hours, 72 hours, and 96 hours respectively. The total RNA of the cells was extracted using TRIzol reagent and reverse transcribed into cDNA. Using cDNA as a template, fluorescence quantitative PCR was performed with the primer pairs CrtE-F / R, CrtB-F / R, CrtI-F / R, CrtY-F / R, CrtZ-F / R, and CrtW-F / R in Table 1 to analyze the expression levels of genes related to carotenoid synthesis (crtE, crtB, crtI, crtY, crtZ, 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 procedure: UDG activation at 50°C for 2 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing / extension at 60°C for 30 s, 40 cycles, and fluorescence signal collection after each cycle.

[0151] After the reaction, the Ct (Cycles threshold) value was obtained, and the data was analyzed and processed, and the relative expression levels of each gene were calculated using 2 -△△Ct calculation formula.

[0152] The results of RT-qPCR analysis were as Figure 5 shown: At 48 hours of culture, the expressions of crtE and crtB genes were significantly up-regulated, promoting the efficient synthesis of phytoene; at 96 hours of culture, the expressions of crtI, crtY, crtW, and crtZ genes gradually increased, which were related to the synthesis of other carotenoids (such as astaxanthin). The carotenoid synthesis pathway was as Figure 6 shown.

[0153] Example 3. Animal safety evaluation of Brevundimonas sp. Bv-xms2024

[0154] 1. Mouse safety evaluation

[0155] Brevundimonas sp. Bv-xms2024 was inoculated into liquid LB medium and cultured at 25°C and 170 rpm for 96 hours. The culture solution was centrifuged at 5000 rpm for 10 minutes to collect the bacterial cells. The bacterial cells were suspended in sterile normal saline to prepare Bv-xms2024 bacterial suspensions with concentrations of 1 × 10 9 CFU / mL, 1 × 10 10 CFU / mL, and 1 × 10 11 CFU / mL.

[0156] Forty healthy Kunming mice were selected and randomly divided into 4 groups, with 10 mice in each group, half male and half female. The healthy Kunming mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The following treatments were performed on the 4 groups of mice respectively:

[0157] Control group: intragastrically administered 0.2 mL of sterile normal saline every day;

[0158] Low-dose group (LG group): intragastrically administered 0.2 mL of Bv-xms2024 bacterial suspension at a concentration of 1 × 10 9 CFU / mL every day;

[0159] Medium-dose group (MG group): intragastrically administered 0.2 mL of Bv-xms2024 bacterial suspension at a concentration of 1 × 10 10 CFU / mL every day;

[0160] High-dose group (HG group): intragastrically administered 0.2 mL of Bv-xms2024 bacterial suspension at a concentration of 1 × 10 11 CFU / mL every day.

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

[0162] Results: During the entire experimental period, all the mice in each group were healthy and showed no obvious abnormal behaviors (such as hair erection or decreased appetite). As shown in Table 2, there was no significant difference in the weight gain trend between the control group and each dose group (P>0.05). Therefore, oral administration of the Bv-xms2024 strain had no toxic effect on mice.

[0163] Table 2

[0164]

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

[0166] 2. Safety evaluation in chickens

[0167] Brevundimonas sp. Bv-xms2024 was 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 selected for the experiment and randomly divided into a control group and an experimental group, with 10 chickens in each group. The SPF chickens were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. The chickens in the control group and the experimental group were treated as follows:

[0169] Control group: drank normal drinking water every day;

[0170] Experimental group: drank the Bv-xms2024 bacterial suspension after culturing in LB for 96 hours every day as a substitute for drinking water.

[0171] Before the experiment, the chickens underwent a 1-week adaptation period. The experimental period was 14 days, and the body weight and health status were recorded every 3 days.

[0172] Results:

[0173] During the entire experimental period, no health problems occurred in all chickens, and there were no deaths or disease events. As shown in Table 3, the body weight gain trends of the chicks in the control group and the experimental group were similar, with no significant difference (P>0.05). The chicken flock maintained normal feeding and activities, indicating that the drinking method of Bv-xms2024 had no obvious toxic effect on poultry.

[0174] Table 3

[0175]

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

[0177] The above experiment shows that Brevundimonas sp. Bv-xms2024 and its fermentation products, as feed additives, have no adverse effects on the health status of mice and poultry and have good safety.

Claims

1. A Brevundimonas vesicularis strain producing phytoene, whose deposit number is CGMCC No.30441.

2. A culture of the Brevundimonas vesicularis strain according to claim 1.

3. A bacterial agent comprising the Brevundimonas vesicularis strain according to claim 1.

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

5. A feed additive comprising the Brevundimonas vesicularis strain according to claim 1.

6. The feed additive according to claim 5, characterized in that The feed additive is the bacterial cell 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 state.

8. Use of the Brevundimonas vesicularis strain according to claim 1 in the preparation of livestock and poultry feed additives.

9. Use of the Brevundimonas vesicularis strain according to claim 1 in producing phytoene.

10. A method for producing phytoene, comprising: The Brevundimonas vesicularis strain according to claim 1 is inoculated into a liquid culture medium and cultured at 20-30° C. and 150-170 rpm for 72-96 hours.

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