Flavobacterium sp. SCSIO 12754 with heme producing function and application of flavobacterium sp. SCSIO 12754

Abstract

The invention discloses a flavobacterium sp. SCSIO 12754 strain with a heme producing function and an application of the flavobacterium sp. SCSIO 12754 strain. The bacterial strain is Corallicola sp.SCSIO 12754, the bacterial strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2024, the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.32944. The invention further discloses a preparation method of the bacterial strain. The Corallicola sp.SCSIO 12754 disclosed by the invention is a new species of flavobacteria, can be used for producing the heme, and can provide an excellent strain resource for the microbial synthesis of the heme.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a strain of Flavobacterium SCSIO 12754 with heme-producing function and application thereof. Background Art

[0002] Heme, formed by the complexation of protoporphyrin IX (PPIX) and ferrous ions, is widely present in various organisms in nature. Heme is involved in maintaining the supply and transport of iron in the body and is an important component of related proteins in respiration and the electron transport chain. In addition, heme also plays an important role in regulating circadian rhythms, signal transduction, gene expression regulation, miRNA processing and synthesis, and other life activities. With the deepening of various studies, the function of heme will be further explored.

[0003] Based on its structure and multiple functions, heme has a wide range of uses in practical applications: since heme iron is more easily absorbed, it can be used as an iron supplement and anti-anemia drug in medical care; in the food industry, heme can replace nitrites and artificial synthetic pigments that are carcinogenic to the human body, increasing food safety; in disease diagnosis and treatment, heme is widely used to treat iron deficiency, acute intermittent porphyria, malarial parasite lysis, etc., and can also be used to prepare porphyrin series drugs in anti-cancer drugs.

[0004] At present, the preparation methods of heme mainly include glacial acetic acid method, acid acetone method (sodium acid method, distillation method, tannic acid method, etc.), carboxymethyl cellulose (CMC) method and protease hydrolysis method, etc., which have the problems of low product purity, high extraction cost and long production cycle. Therefore, biological fermentation method has attracted attention, among which the production of heme by Escherichia coli has been realized. However, Escherichia coli has the limitations of high endotoxin content and poor genetic stability of plasmid as a host. Therefore, developing a new host suitable for industrial application to replace Escherichia coli to produce heme has become the focus and direction of current research. Summary of the invention

[0005] The first object of the present invention is to provide a new Flavobacterium (Coralliicola sp.) SCSIO 12754, which was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 6, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32944.

[0006] The Flavobacterium SCSIO 12754 of the present invention is isolated from coral sample tissue collected from the Luhuitou sea area of ​​Sanya (109°29′E, 18°13′N).

[0007] The isolated and purified new species of Flavobacterium SCSIO 12754 was streaked into MA medium and cultured at 25°C for 3 days. The morphological characteristics of the strain were observed. The colonies were milky white, round, and smooth and moist ( Figure 1 ).

[0008] The suitable growth temperature range of the Flavobacterium SCSIO 12754 is 15°C to 35°C, the pH range is 5 to 8, and the salinity range is 1% to 4% NaCl by mass percentage.

[0009] Preferably, the most suitable growth temperature of the Flavobacterium SCSIO 12754 is 30°C, the pH range is 6-7, and the salinity range is 3% NaCl by mass percentage.

[0010] The 16S rRNA sequence of the new microbial strain Coralliicola sp.SCSIO 12754 is shown in SEQ ID NO.1, specifically:

[0011] ATGAAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAACACATGCAAGT

[0012] CGAACGGTAACAGGAAAGAGCTTGCTCTTTTGCTGACGAGTGGCGCACGGGTGCGTAA

[0013] CGCGTATGCAATCTACCTTGTACAGGGGGATAGCCCAGAGAAATTTGGATTAATATCCCAT

[0014] AGTATTATTGATTGGCATCGATTAATAATTAAAGCTTCGGTGGTACAAGATGAGCATGCGT

[0015] CTTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCAACGATAAGTAGGGGCCCTGAG

[0016] AGGGGGATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAG

[0017] TGAGGAATATTGGACAATGGTCGAAAGACTGATCCAGCCATGCCGCGTGCAGGAAGACT

[0018] GCCCTATGGGTTGTAAACTGCTTTTATACAGGAAGAATAAGGATCTCGTGAGGTCTGATG

[0019] ACGGTACTGTAAGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG

[0020] GGTGCAAGCGTTATCCGGAATCATTGGGTTTAAAGGGTCCGTAGGCGGGCAAGTAAGTC

[0021] AGAGGTGAAAGTTTGCGGCTCAACCGTAAAATTGCCTTTGATACTGCTAGTCTTGAATAT

[0022] GTGTGAAGTGGTTAGAATAAGTAGTGTAGCGGTGAAATGCATAGAGATTACTTAGAATAC

[0023] CGATTGCGAAGGCAGATCACTAACACATTATTGACGCTGATGGACGAAAGCGTGGGGAG

[0024] CGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGGATACTAGCTGTTTGAA

[0025] CTTCGGTTTGAGTGGCTAAGCGAAAGTGATAAGTATCCCACCTGGGGAGTACGTTCGCA

[0026] AGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA

[0027] ATTCGATGATACGCGAGGAACCTTACCAAGGCTTAAATGTAGTATGACAGGTTTAGAGAT

[0028] AGACTTTTCTTCGGACATATTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAG

[0029] GTGTCAGGTTAAGTCCTATAACGAGCGCAACCCCTGTTGTTAGTTACCAGCACGTAAAG

[0030] GTGGGGACTCTAGCAAGACTGCCGGTGCAAACCGTGAGGAAGGTGGGGATGACGTCAA

[0031] ATCATCACGGCCCTTACGCCTTGGGCTACACACGTGCTACAATGGTAGGTACAGAGAGC

[0032] AGCCATCTGGTGACAGAGAGCGAATCTACAAAACCTATCTCAGTTCGGATCGGAGTCTG

[0033] CAACTCGACTCCGTGAAGCTGGAATCGCTAGTAATCGGATATCAGCCATGATCCGGTGAA

[0034] TACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGCTGGGGGTACCTGAA

[0035] GTCGGTGACCGTAAGGAGCTGCCTAGGGTAAAACTGGTAACTGGGGCTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGAACACCTCCTT.

[0036] Blast comparison analysis of the 16S rRNA gene sequence of the microbial strain revealed that it was similar to the strain Lutaonella thermophila CC-MHSW-2 T The sequence similarity of the strain SCSIO 12754 was the highest, at 93.85%. The microbial strain SCSIO 12754 was a member of the Flavobacteriaceae family. The phylogenetic tree was constructed using the neighbor-joining method, which clearly reflected the relationship between the strain and the members of the Flavobacteriaceae family. Figure 2 ). Through the above morphological and molecular biological identification, it was determined that SCSIO 12754 should be a new genus and species of Flavobacteriaceae, and was named Coralliicolasp.SCSIO 12754.

[0037] The second object of the present invention is to provide the use of the Coralliicola sp. SCSIO 12754 in producing heme.

[0038] The invention also provides a method for producing hemoglobin, which comprises inoculating Flavobacterium Coralliicola sp.SCSIO12754 into a fermentation medium to produce hemoglobin through fermentation.

[0039] Preferably, the formula of the fermentation medium is: 5g peptone, 1g yeast extract, 0.1g ferric citrate per liter of seawater, pH 7.2-7.4.

[0040] The invention provides a new Flavobacterium (Coralliicola sp.) SCSIO 12754, which can provide a large amount of heme by fermentation and can provide excellent strain resources for microbial synthesis of heme.

[0041] Coralliicola sp.SCSIO 12754, which was deposited on December 6, 2024 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the deposit number is CGMCC No.32944. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The colony morphology of the strain SCSIO 12754 of the present invention was cultured on MA medium at 25°C for 3 days.

[0043] Figure 2 The phylogenetic tree of strain SCSIO 12754 and strains with high sequence similarity in the same family and other strains constructed based on the 16S rRNA gene sequence, wherein SCSIO 12754 is the strain used in the present invention.

[0044] Figure 3 is the standard curve of hemoglobin. DETAILED DESCRIPTION

[0045] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0046] Example 1 Isolation and culture of strain SCSIO 12754

[0047] Coral samples were collected from the Luhuitou reef area in Sanya (109°29′E, 18°13′N). The surface of the coral samples was washed with sterile seawater and then a 5 cm 3The coral tissue was washed with an air gun to obtain a coral tissue suspension. A 10-fold dilution of the tissue suspension was made with sterile seawater, and 100 μL of each dilution was evenly spread on MA medium (the formula of MA medium is: 5g peptone, 1g yeast extract, 0.1g ferric citrate, 15g agar powder, 1L seawater, pH 7.2-7.4). After culturing at 25°C for 7 days, a single colony was picked and streaked on MA medium for purification. The purified strain was preserved at -80°C using glycerol (25%, w / v) preservation method, thereby obtaining strain SCSIO 12754.

[0048] Example 2 Identification of strain SCSIO 12754

[0049] The genomic DNA of strain SCSIO 12754 was extracted and PCR amplified using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') of bacterial 16S rRNA gene. The amplification primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and Taq enzyme was purchased from TaKaRa. The amplification system of 16S rRNA gene was as follows: forward primer 27F 2μL, reverse primer 1492R 2μL, DNA template 1μL, Taq enzyme mixture 25μL, and water was added to make up to 50μL. The PCR reaction program was: 95℃5min; 94℃30s, 55℃30min, 72℃1min 30s (30cycles), 72℃5min. The PCR amplification products were analyzed by electrophoresis on 1.0% (W / V) agarose gel, and the experimental results were detected by gel imaging. Positive results were sent to Shanghai Biotech Co., Ltd. for sequencing.

[0050] After obtaining the 16S rRNA gene sequence data of strain SCSIO 12754, the BLAST software GenBank / EMBL / DDBJ database and EzBioCloud database (https: / / www.ezbiocloud.net / ) were used to perform online sequence comparison. The comparison results showed that the strain SCSIO 12754 of the present invention is a member of the Flavobacteriaceae family and is similar to the strain Lutaonella thermophila CC-MHSW-2 T (sequence number EU287913) has the highest sequence similarity of 93.85%, which is lower than the 95% threshold for defining different bacterial genera. In order to further clarify the phylogenetic position of the strain, representative strain sequences with high similarity to the 16S rRNA gene sequence of the strain of the present invention were selected to construct the NJ phylogenetic tree, such as Figure 2As shown, the strain SCSIO 12754 was identified as a new genus and species of Flavobacteriaceae. The bacterium was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on December 6, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 32944.

[0051] Example 3 Cultivation of the strain Coralliicola sp. SCSIO 12754 of the present invention and analysis of heme production

[0052] The strain SCSIO 12754 was inoculated into 5 mL MB liquid medium and cultured at 25°C and 180 rpm for 3 days until the end of the logarithmic growth phase. Subsequently, the bacterial solution was transferred to 50 mL fresh MB liquid medium (formula: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 1 L seawater, pH 7.2-7.4) at a 1% inoculation ratio and continued to be cultured at 25°C and 180 rpm for 3 days. After the culture was completed, the culture solution was centrifuged at 4°C and 12000 r / min for 5 minutes to collect the bacterial precipitate. The bacterial cells were resuspended with 10 mL sterile deionized water and centrifuged again at 12000 r / min for 5 minutes. The supernatant was discarded and the wet weight of the bacterial cells was recorded. 0.38 g of bacterial cells were resuspended with 500 μL of 20 mmol / L oxalic acid solution, and then transferred to a 1.5 mL amber centrifuge tube, gently mixed and allowed to stand at 4°C for 16 hours. Then add 500 microliters of 2 mol / L oxalic acid solution to the centrifuge tube and mix well. Then take 500 microliters of sample and transfer it to another 1.5 ml amber centrifuge tube. Heat it in a 95℃ water bath for 30 minutes to react. It is used to detect the fluorescence value of the total concentration of porphyrin and hemoglobin. Another 500 microliters of sample is placed at room temperature for the detection of the fluorescence value of porphyrin concentration. After the sample is cooled to room temperature, centrifuge it at 12000 rpm for 5 minutes. Take 200 microliters of supernatant and measure the fluorescence value in a 96-well plate with a black transparent bottom. The detection conditions are an excitation wavelength of 400nm and an emission wavelength of 620nm. The difference in the fluorescence values ​​of the above two groups is calculated as the fluorescence value corresponding to the hemoglobin concentration to be measured.

[0053] Use 0.25% (w / v) Na 2 CO 3The solution is prepared with different concentrations of hemin chloride standard samples, and the standard samples of different concentrations are mixed with 2 mol / L oxalic acid solution in equal volumes of 1:1, and then placed in a 95°C water bath for 30 minutes to react. After the standard sample cools to room temperature, take 200 microliters of the supernatant and measure the fluorescence value in a 96-well plate with a black transparent bottom. The detection conditions are an excitation wavelength of 400nm and an emission wavelength of 620nm. Draw a standard curve based on the fluorescence value and hemin concentration, with the fluorescence value as the ordinate and the hemin concentration as the abscissa. Draw the standard curve as shown in the figure. Figure 3 .

[0054] The fluorescence difference obtained from the above experimental sample detection was used as the y value, and substituted into the formula y=2385.9x+10139 to calculate the x value, which is the heme concentration in the experimental sample. Then, based on the wet weight of the cells used for heme extraction, the heme content per gram of wet cells was calculated, and the result was 98.67±5.32 micrograms / gram of wet cells.

[0055] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Coralliicola sp. SCSIO 12754, whose deposit number is: CGMCC No.32944.

2. A microbial agent, characterized in that: The invention contains the Flavobacterium Coralliicolasp. SCSIO 12754 as claimed in claim 1 as an active ingredient.

3. Use of the Flavobacterium Coralliicola sp.SCSIO 12754 described in claim 1 in producing heme.

4. A method for producing hemoglobin, characterized in that The method comprises inoculating the Flavobacterium Coralliicolasp. SCSIO 12754 described in claim 1 into a fermentation medium to produce heme by fermentation.

5. The method according to claim 4, characterized in that The formula of the fermentation medium is that per liter of seawater, it contains: 5g of peptone, 1g of yeast extract, 0.1g of ferric citrate, and pH 7.2-7.4.

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