SHEWANELLA DECOLORATIONIS PRODUCING TETRODOTOXIN AND APPLICATION THEREOF

Abstract

Disclosed are a Shewanella decolorationis producing tetrodotoxin and an application thereof, falling in the field of development and utilization of medicinal microorganisms. A strain, Shewanella decolorationis S3-4, is deposited in the China General Microbiological Culture Collection Center (CGMCC) on Mar. 28, 2022, with a deposit number of CGMCC No. 24602. The strain can secrete a same substance as tetrodotoxin from Tetraodontidae.

Claims

1. A Shewanella decolorationis producing tetrodotoxin, a strain of S3-4, being classified as Shewanella decolorationis and deposited in the China General Microbiological Culture Collection Center (CGMCC) on Mar. 28, 2022 with a deposit number of CGMCC No. 24602.

2. A method for preparing tetrodotoxin by utilizing the Shewanella decolorationis according to claim 1, comprising, inoculating Shewanella decolorationis S3-4 into an LB liquid culture medium, and culturing a same at 28? ? C. at 200 rpm for 2-3 days.

3. The method according to claim 2, wherein the LB liquid culture medium comprises the following components: tryptone with a final concentration of 10.0 g/L, yeast extract powder with a final concentration of 5.0 g/L, and sodium chloride with a final concentration of 10.0 g/L; and the strain of Shewanella decolorationis S3-4 obtained by fermentation culture in the LB liquid culture medium is crushed, isolated and purified to obtain tetrodotoxin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a bacterial identification diagram of a full-length 16SrDNA of Shewanella decolorationis S3-4 bacterium.

[0016] FIG. 2 is an ion chromatography-mass spectrometry diagram of a tetrodotoxin standard substance (0.5 ng/ml).

[0017] FIG. 3 is a graph of detection standard.

[0018] FIG. 4 is a liquid mass spectrum diagram of a bacterial source sample of Shewanella decolorationis S3-4.

DETAILED DESCRIPTION

[0019] Technical solutions of the present disclosure will be further explained by the following examples, but the protection scope of the present disclosure is not limited by the examples in any form.

Example 1

[0020] Culture media used in the example included: 2216E solid culture media, TCBS solid culture media, LB solid culture media and LB liquid culture media, which were all commercial culture media and purchased from Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd.

[0021] Takifugu ocellatus is a small warm-water carnivorous bottom fish, which mainly inhabites nearshore waters and sometimes enters freshwater rivers and brackish-freshwater estuaries. The Takifugu ocellatus has an air bag, which could make its abdomen swell for self-defense when it is attacked by an enemy. The Takifugu ocellatus preys on small shellfish, crablat, amphipods, small crayfish and algae debris. When entering fresh water, the Takifugu ocellatus preys on shrimps, crabs, mussels, fry, aquatic insect larvae, angular and copepods, and occasionally aquatic vascular plants and filamentous algae. During the spawning period from April to June, fish schools migrate to the middle and lower reaches of rivers to spawn, laying demersal and viscid eggs, which belongs to one-time spawning type. The Takifugu ocellatus is hypertoxic in ovary and liver, non-toxic in testis, highly toxic in skins and intestines, and weakly toxic in muscle. In the present disclosure, the ovary, liver and intestines of the Takifugu ocellatus with strong toxicity were selected for research. [0022] (1) A wild Takifugu ocellatus was selected and collected by the inventor from the estuary of Yifeng River in Shantou City, Guangdong Province. Ovarian tissues, liver and intestinal tissues of Takifugu ocellatus containing tetrodotoxin (TTX) were taken. [0023] (2) Tissue homogenate was performed, and diluted solution was prepared with PBS at volume-mass ratios of 1:10, 1:100 and 1:1000. 100 ?l of the diluted solution was taken and coated on the 2216E solid culture medium, TCBS solid culture medium and LB solid culture medium. [0024] (3) After culturing at 28? C. for 48 hours, characteristic colonies on a plate were picked out and streaked on another 2216E solid culture medium, TCBS solid culture medium and LB solid culture medium. [0025] (4) After culturing at 28?C for 24 hours, an isolated single colony was streaked again for isolated culture. [0026] (5) Streaking culture was repeated until the single colony was isolated.

[0027] The tissues in step (1) were the liver, ovary and intestines of the Takifugu ocellatus.

[0028] The 2216E solid culture medium included the following components: 5.0 g/L of peptone, 1.0 g/L of yeast powder, 0.1 g/L of ferric citrate, 19.45 g/L of sodium chloride, 5.98 g/L of magnesium chloride, 3.24 g/L of sodium sulfate, 1.8 g/L of calcium chloride, 0.55 g/L of potassium chloride, 0.16 g/L of sodium carbonate, 0.08 g/L of potassium bromide, 0.034 g/L of strontium chloride, 0.022 g/L of boric acid, 0.004 g/L of sodium silicate, 0.0024 g/L of sodium fluoride, 0.0016 g/L of ammonium nitrate, 0.008 g/L of disodium hydrogen phosphate and 15.0 g/L of agar.

[0029] The TCBS solid culture medium included the following components: 5.0 g/L of yeast extract powder, 10.0 g/L of peptone, 10.0 g/L of sodium thiosulfate, 10.0 g/L of sodium citrate, 5.0 g/L of Ox gallbladder powder, 3.0 g/L of natrii tauroglycocholas, 20.0 g/L of sucrose, 10.0 g/L of sodium chloride and 1.0 g/L of iron citrate, 0.04 g/L of bromothymol blue, 0.04 g/L of thymol blue and 15.0 g/L of agar.

[0030] The LB liquid culture medium included the following components: 10.0 g/L of tryptone, 5.0 g/L of yeast extract powder, 10.0 g/L of sodium chloride and 15.0 g/L of agar.

[0031] A strain was inoculated into the LB liquid culture medium, and cultured at 28? C. and 200 rpm for 2-3 days. A fermentation broth was detected by liquid chromatography-mass spectrometry, and it was found that a strain S3-4 could produce tetrodotoxin. The strain had the following morphological, physiological and biochemical characteristics.

(1) Morphological Characteristics of Bacteria

[0032] According to the conventional physiological and biochemical identification methods of bacteria, cells of Shewanella decolorationis were Gram-negative bacteria, with a rod-shape and a size of about (0.6-1.0)?(1.0-6.7) ?m, having cilia all over the body and a single polar flagellum.

(2) Main Physiological and Biochemical Characteristics

[0033] The strain had ability of facultative anaerobic growth, reducing trivalent iron, liquefying gelatin, and producing H2S. The oxidase and contact enzyme were positive, and could grow with sodium lactate, D-galactose and succinic acid as the only carbon source, but could not grow with ammonium sulfate and ammonium nitrate as the only nitrogen source.

(3) A Method for Identifying Bacteria by a 16SrDNA Full-Length Sequence:

[0034] the DNA of the genome of the strain was obtained by extraction and isolation, 16SrDNA was amplified by PCR, and an upstream primer: 5-agagtttgatcctggctcag-3, a downstream primer: 5-tacgacttaaccccaatccgc-3, and 50 ?l of a PCR reaction system were used. The PCR reaction system included: 25 ?l of 2?Rapid Taq Master Mix, 1 ?l of the upstream primer, 1 ?l of the downstream primer, and 50 ng of the bacterial genomic DNA.

[0035] The PCR reaction program was run at 95? C. for 3 minutes; 95? ? C. for 15 seconds, 55? C. for 15 seconds and 72?C for 30 seconds, totally 35 cycles; and 72?C for 5 minutes. The amplified product of 16S rDNA full-length sequence was purified and recovered, and the recovered product was cloned in a Tl vector. Escherichia coli was transformed; positive clones were selected for sequencing, sequencing results were compared by BLAST N in GenBank, and the sequences with high homology were obtained. A MEGA X software was utilized for cluster analysis to determine that the isolated strain was Shewanella decolorationis S3-4, and the results were shown in FIG. 1. The isolated strain was Shewanella decolorationis S3-4.

[0036] 16sDNA sequence of Shewanella decolorationis S3-4 were as follows.

TABLE-US-00001 ggcgcgcggctacacatgcagtcgagcggcagcacaagtgagtttactc atgaggtggcgagcggcggacgggtgagtaatgcctagggatctgccca gtcgagggggataacagttggaaacgactgctaataccgcatacgccct acgggggaaaggaggggaccttttggccttccgcgattggatgaaccta ggtgggattagctagttggtgaggtaatggctcaccaaggcgacgatcc ctagctgttctgagaggatgatcagccacactgggactgagacacggcc cagactcctacgggaggcagcagtggggaatattgcacaatgggggaaa ccctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaa gcactttcagtagggaggaaaggttgtaagttaataccttgcagctgtg acgttacctacagaagaaggaccggctaactccgtgccagcagccgcgg taatacggagggtccaagcgttaatcggaattactgggcgtaaagcgtg cgcaggcggtttgttaagcgagatgtgaaagccccgggctcaacctggg aattgcatttcgaactggcaaactagagtcttgtagaggggggtagaat tccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggc gaaggcggccccctggacaaagactgacgctcaggcacgaaagcgtggg gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtct actcggagtttggtgtcttgaacactgggctctcaagctaacgcattaa gtagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattg acgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacg cgaagaaccttacctactcttgacatccagagaactttccagagatgga ttggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagc tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccctat ccttatttgccagcgcgtaatggcgggaactctagggagactgccggtg ataaaccggaggaaggtggggacgacgtcaagtcatcatggcccttacg agtagggctacacacgtgctacaatggcgagtacagagggttgcaaagc cgcgaggtggagctaatctcacaaagctcgtcgtagtccggattggagt ctgcaactcgactccatgaagtcggaatcgctagtaatcgtgaatcaga atgtcacggtgaatacgttcccgggccttgtacacaccgcccgtcacac catgggagtgggctgcaaaagaagtgggtagcttaacctcgggagggcg c.

[0037] A Shewanella decolorationis producing tetrodotoxin, a strain of S3-4, was deposited in the China General Microbiological Culture Collection Center (CGMCC). The deposit address was No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing. The preservation date was Mar. 28, 2022. The deposit number was CGMCC No. 24602.

Example 2

[0038] A fermentation culture method for Shewanella decolorationis S3-4 isolated in Example 1 included the following steps. [0039] (1) Bacteria were inoculated into an LB liquid culture medium. [0040] (2) The LB liquid culture medium was cultured at 28? C. at 200 rpm for 2-3 days.

[0041] The LB liquid culture medium included the following components: 10.0 g/L of tryptone, 5.0 g/L of yeast extract powder and 10.0 g/L of sodium chloride.

Example 3

[0042] A preparation for tetrodotoxin by utilizing the fermentation of Shewanella decolorationis S3-4 obtained in Example 2 included the following specific steps. [0043] (1) Shewanella decolorationis S3-4 was fermented and cultured, and then centrifuged at 4?C at 4000?g for 30 minutes to collect strain cells. [0044] (2) 0.1% methanol formate was added and cells were ultrasonically crushed. [0045] (3) The cells were centrifuged at 8000 rpm for 5 minutes. [0046] (4) Supernatant was taken and filtered through an organic filter membrane with a pore size of 0.22 ?m. [0047] (5) Tetrodotoxin was detected by liquid chromatography-mass spectrometry. [0048] (6) Liquid chromatography-mass spectrums of a tetrodotoxin standard substance were shown in FIG. 2. [0049] (7) A result of a standard curve was shown in FIG. 3. [0050] (8) Results of liquid chromatography-mass spectrometry for the identification of Shewanella decolorationis-derived tetrodotoxin were shown in FIG. 4, the liquid chromatography-mass spectrometry of the Shewanella decolorationis-derived tetrodotoxin being consistent with that of the standard substance.

[0051] In an example of the present disclosure, bacteria were isolated from the ovary, liver and intestinal tissues of Takifugu ocellatus, and screened using the 2216E solid culture medium, TCBS solid culture medium and LB solid culture medium. A Shewanella decolorationis S3-4 capable of producing tetrodotoxin was isolated from the Tetraodontidae body for the first time, and the isolated bacteria were identified by 16S rDNA method.

[0052] The tetrodotoxin produced by the isolated Shewanella decolorationis S3-4 was detected by a liquid chromatography-mass spectrometry (LC-MS) method. The detection on a compound of tetrodotoxin by an LC-MS instrument is more accurate. The tetrodotoxin produced by Shewanella decolorationis S3-4 was determined to be a same substance as the tetrodotoxin extracted from Tetraodontidae.

[0053] According to the present disclosure, the isolated Shewanella decolorationis S3-4 can be cultured to isolate tetrodotoxin from bacteria on a large scale.

[0054] The basic principle, main features and advantages of the present disclosure have been shown and described above. It is to be understood by those skilled in the art that the present disclosure is not limited by the above examples, and what is described in the above examples and specifications only illustrates the principles of the present disclosure, and there will be various changes and improvements in the present disclosure without departing from the spirit and scope of the present disclosure, which fall within the scope of the claimed disclosure. The scope of protection required by the present disclosure is defined by the appended claims and equivalents thereof.