Ectoine-producing halophilic bacteria as well as screening method and application thereof

Abstract

The invention relates to an Ectoine-producing halophilic bacterium as well as a screening method and application thereof, and belongs to the technical field of microorganisms. According to the Ectoin-producing halophilic bacteria disclosed by the invention, the Ectoin-producing halophilic bacteria are Vreelandelassp., and the Ectoin-producing halophilic bacteria are Ectoin-producing halophilic bacteria. The 16S rRNA of the Ectoin-producing halophilic bacteria has at least 90% of nucleotide sequence consistency with that of SEQ ID NO. 1; the Ectoin halophilic bacteria are used for expressing 2-aminobutyric acid acetyltransferase, 2-aminobutyric acid transaminase and Ectoin synthetase. Through an efficient screening method and deep genome analysis, the novel halophilic strain capable of synthesizing the Ectoine is successfully screened out, and the gene cluster for synthesizing the Ectoine is excavated, so that the production efficiency and yield of the Ectoine can be improved, a microbial resource library is enriched, and the genetic diversity is increased.

Description

A halophilic bacterium producing ectoine and its screening method and application Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an ectoin-producing halophilic bacterium and its screening method and application. Background Technology

[0002] Ectoine is a zwitterionic amino acid derivative with strong hydrophilicity and water molecule complexation ability, forming an "Ectoine hydroelectric complex." This complex surrounds biomolecules such as cells, enzymes, and proteins, constructing a protective, nourishing, and stable hydration shell to maintain cellular osmotic pressure balance and protect intracellular proteins, enzymes, and nucleic acids from damage caused by extreme conditions. Discovering new ectoine-producing strains and their biosynthetic gene clusters can enrich the microbial resource library and provide new samples and data support for biodiversity research. These discoveries not only provide new tools and targets for synthetic biology and genetic engineering but also optimize ectoine production efficiency. Ectoine has broad application prospects in the pharmaceutical and cosmetic fields, and can be used to develop anti-aging, moisturizing, and anti-UV products. Furthermore, introducing ectoine biosynthetic genes into plants through genetic engineering can enhance their survival ability under drought and saline-alkali conditions, thereby contributing to the protection and restoration of ecosystems around salt lakes.

[0003] With increasingly severe environmental challenges, the discovery of new ectoine-producing strains is of paramount importance. Currently, ectoine production mainly relies on genetically engineered strains. However, with the intensification of climate change, existing technologies still have some limitations: on the one hand, halophilic bacteria can produce more protective ectoines through mutation in extreme environments, but current research on these mutation mechanisms is not in-depth enough, and they have not been fully explored and effectively utilized, making it difficult to meet the needs of increasingly diverse application scenarios; on the other hand, the existing strains used for ectoine production lack sufficient genetic diversity, which to some extent limits their wide applicability in different application scenarios and makes it difficult to fully adapt to diverse market demands.

[0004] Therefore, there is an urgent need to develop more ectoine-producing microorganisms. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ectoin-producing halophilic bacterium, its screening method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for screening ectoine-producing halophilic bacteria, comprising the following steps: S1, adding the collected sample to a high-salt culture medium and culturing for 3-5 days to obtain a fermentation broth, and using high-performance liquid chromatography to detect the ectoine content in the fermentation broth to screen for a mixed ectoine-producing bacterial community; S2, inoculating the mixed ectoine-producing bacterial community obtained in step S1 into a high-salt culture medium with NaCl concentration increasing by 1 wt% and performing continuous subculturing at least 5 times to obtain enriched ectoine-producing halophilic bacteria; S3, isolating and purifying the enriched ectoine-producing halophilic bacteria obtained in step S2 to obtain purified ectoine-producing halophilic bacteria.

[0007] This invention involves inoculating suspected ectoine-producing microorganisms into a high-salt culture medium and culturing them. High-performance liquid chromatography (HPLC) is used to detect the presence of ectoine in the fermentation broth to screen for a mixed ectoine-producing bacterial community. Further screening and enrichment of ectoine-producing halophilic bacteria are then achieved using a gradient concentration of high-salt culture medium. Finally, the ectoine-producing halophilic bacteria are isolated and purified. This screening method effectively screens for and enriches ectoine-producing halophilic bacteria.

[0008] In a preferred embodiment of the screening method of the present invention, in step S1, the high-salt culture medium is a culture medium containing at least 3 wt% NaCl. In the present invention, different types of culture media can be selected according to the source of the sample or the screening purpose, and at least 3 wt% NaCl can be added to the culture medium to achieve the purpose of screening halophilic bacteria.

[0009] In a preferred embodiment of the screening method of the present invention, in step S1, the culture conditions are 28-32℃, 180-220rpm and 2-4 days.

[0010] In a preferred embodiment of the screening method of the present invention, in step S1, the culture conditions are 30°C, 200 rpm and 3 days.

[0011] In a preferred embodiment of the screening method of the present invention, in step S2, the high-salt culture medium with NaCl concentration increasing by 1 wt% is characterized by a NaCl concentration of 3 wt% at the initial culture, 4 wt% at the first subculture, 5 wt% at the second subculture, 6 wt% at the third subculture, 7 wt% at the fourth subculture, and 8 wt% at the fifth subculture, with the NaCl concentration in the high-salt culture medium increasing by 1 wt% with each subculture.

[0012] As a preferred embodiment of the screening method of the present invention, in step S2, the culture conditions for continuous subculturing are 28-32℃, 180-220rpm and 6-8 days.

[0013] As a preferred embodiment of the screening method of the present invention, in step S2, the culture conditions for continuous subculturing are 30°C, 200 rpm and 7 days.

[0014] In a preferred embodiment of the screening method described in this invention, in step S3, the separation and purification involves diluting the enriched ectoin-producing halophilic bacteria in a 10-fold serial gradient, and taking 10% of the diluted product... -4 10 -5 10 -6 and 10 -7 The bacterial suspension was spread onto high-salt solid medium plates and cultured for 6-8 days. Single colonies were selected and inoculated onto new high-salt medium plates for subculturing for at least 3 generations to obtain purified ectoin-producing halophilic bacteria.

[0015] As a preferred embodiment of the screening method of the present invention, the screening method further includes the following steps: S4, the purified ectoin-producing halophilic bacteria obtained in step S3 are inoculated into a high-salt culture medium containing 7wt% NaCl to obtain a bacterial solution, and the ectoin content of the bacterial solution is detected by HPLC to screen for high ectoin-producing halophilic bacteria.

[0016] Secondly, the present invention provides an ectoine-producing halophilic bacterium, wherein the ectoine-producing halophilic bacterium is Vreelandella sp.; the 16S rRNA of the ectoine-producing halophilic bacterium has at least 90% nucleotide sequence identity with SEQ ID NO.1; the ectoine-producing halophilic bacterium expresses 2-aminobutyric acid acetyltransferase, 2-aminobutyric acid transaminase and ectoine synthase.

[0017] This invention, through efficient screening methods and in-depth genomic analysis, successfully screened novel halophilic bacterial strains capable of synthesizing ectoine and identified their ectoine-synthesizing gene clusters. This not only improves ectoine production efficiency and yield but also enriches the microbial resource library and increases genetic diversity. A thorough understanding of the strain's characteristics, including physiological and biochemical properties, chemical composition, and GC content, provides a solid theoretical foundation for subsequent research and applications. These advantages collectively ensure that this invention can meet the diverse application needs of fields such as medicine, cosmetics, and agriculture, and has broad application prospects.

[0018] As a preferred embodiment of the ectoin-producing halophilic bacterium of the present invention, the amino acid sequence of the 2-aminobutyric acid acetyltransferase is shown in SEQ ID NO.2, the amino acid sequence of the 2-aminobutyric acid transaminase is shown in SEQ ID NO.3, and the amino acid sequence of the ectoin synthase is shown in SEQ ID NO.4.

[0019] As a preferred embodiment of the ectoin-producing halophilic bacteria of the present invention, the ectoin-producing halophilic bacteria are mainly obtained by screening using the above-mentioned screening method.

[0020] As a preferred embodiment of the ectoin-producing halophilic bacteria of the present invention, the ectoin-producing halophilic bacteria includes GUYU39.

[0021] As a preferred embodiment of the ectoin-producing halophilic bacterium described in this invention, the ectoin-producing halophilic bacterium GUYU39 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36221, deposit date of October 9, 2025, and classified as Vreelandella sp.

[0022] Thirdly, the present invention provides a gene cluster for synthesizing ectoin, comprising ectA, ectB and ectC; the nucleotide sequence of ectA is shown in SEQ ID NO.5, the nucleotide sequence of ectB is shown in SEQ ID NO.6 and the nucleotide sequence of ectC is shown in SEQ ID NO.7.

[0023] Fourthly, the present invention provides a 2-aminobutyric acid acetyltransferase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0024] Fifthly, the present invention provides a 2-aminobutyric acid transaminase, the amino acid sequence of which is shown in SEQ ID NO.3.

[0025] In a sixth aspect, the present invention provides an ectoin synthase, the amino acid sequence of which is shown in SEQ ID NO.4.

[0026] In a seventh aspect, the present invention provides the application of the above-mentioned ectoin-producing halophilic bacteria, ectoin-synthetic gene clusters, 2-aminobutyric acid acetyltransferase, 2-aminobutyric acid transaminase or ectoin synthase in the production of ectoin.

[0027] Eighthly, the present invention provides a method for producing ectoine, comprising the following steps: inoculating the above-mentioned ectoine-producing halophilic bacteria into a high-salt culture medium for fermentation to obtain a fermentation broth, and purifying the fermentation broth to obtain ectoine.

[0028] Compared with existing technologies, the beneficial effects of this invention are as follows: Through efficient screening methods and in-depth genomic analysis, this invention successfully screened novel halophilic bacterial strains capable of synthesizing ectoine and identified their ectoine-synthesizing gene clusters. This not only improves the production efficiency and yield of ectoine but also enriches the microbial resource library and increases genetic diversity. A thorough understanding of the strain's characteristics from multiple aspects, including physiological and biochemical properties, chemical composition features, and GC content, provides a solid theoretical foundation for subsequent research and applications. These advantages collectively ensure that this invention can meet the diverse application needs of fields such as medicine, cosmetics, and agriculture, and has broad application prospects. Attached Figure Description

[0029] Figure 1 shows the standard curve for ectoin detection by HPLC according to the present invention; Figure 2 shows a single colony and transmission electron micrograph of GUYU39 in Example 2 of the present invention, where the scale bar of A is 1 mm and the scale bar of B is 2.0 μm; Figure 3 shows the phylogenetic tree of GUYU39 in Example 2 of the present invention (constructed by the NJ method); Figure 4 shows the thin-layer chromatography results of GUYU39 in Example 3 of the present invention, where the colorimetric reagent of A is molybdenum phosphate solution, the colorimetric reagent of B is molybdenum phosphate solution, the colorimetric reagent of C is ninhydrin solution, and the colorimetric reagent of D is ammonium molybdate solution. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] Unless otherwise specified, “halophilic bacterium GUYU39”, “ectoin-producing halophilic bacterium GUYU39” and “GUYU39” mentioned in the following examples and effect examples are all ectoin-producing halophilic bacterium GUYU39 with accession number CGMCC No. 36221.

[0032] Unless otherwise specified, all materials and reagents used in the following examples and effect examples are commercially available.

[0033] LB medium: Dissolve 10g tryptone, 5g yeast extract, and 10g sodium chloride in 1000mL distilled water. Adjust the pH to 7.0±0.2 and autoclave at 121℃ for 15min. Cool after sterilization. To prepare a high-salt solid medium, add 18g agar powder to the above medium and sterilize at 121℃ for 30min.

[0034] High-salt culture medium: Dissolve 30g NaCl, 10g tryptone, and 5g yeast extract in 1000mL distilled water. Autoclave at 121℃ for 15min, then cool before use. Add 18g agar powder to the above medium to prepare a high-salt solid culture medium, and sterilize at 121℃ for 30min.

[0035] Marine bacteria agar 2216 medium, purchased from BioWind, catalog number M1040-01.

[0036] The specific operation for detecting ectoin content using high performance liquid chromatography is as follows: (1) The isolated single bacteria were inoculated on a high-salt culture medium plate and cultured in an incubator at 37°C for 24 h. A single clone was then inoculated into a 5 mL LB test tube and cultured overnight at 37°C and 200 rpm. After that, 10% bacterial culture was transferred to 100 mL of high-salt liquid culture medium for culture. After fermentation at 37°C and 200 rpm for 48 h, the ectoin content was detected. (2) 800 μL of fermentation broth was added to 2.4 mL of anhydrous ethanol, mixed and reacted for 30 min, centrifuged at 8000 r / min for 10 min, the supernatant was taken, the solvent was removed by nitrogen blowing, 2.4 mL of anhydrous ethanol was added and dissolved by sonication, the supernatant was taken by centrifugation, nitrogen blowing was added, 800 μL of water was added and shaken to dissolve, and the ectoin content was detected by HPLC. (3) The ectoin mother liquor with a concentration of 0.5 g / L was diluted with ethanol to prepare 5 mg / L, 10 mg / L, 30 mg / L, and 50 mg / L solutions, respectively. A series of standard working solutions of ectoine at concentrations of 5 mg / L and 100 mg / L were filtered through a 0.22 μm filter membrane and detected by HPLC. A standard curve was plotted with the concentration of the ectoine standard working solution on the x-axis and the HPLC peak area of ​​ectoine on the y-axis (see Figure 1). In this detection method, ectoine showed a linear relationship in the concentration range of 5-100 mg / L, and the equation of the standard curve was y = 37.792. x+15.207, correlation coefficient R 2 =0.9999, indicating that the concentration of ectoine in the sample can be quantified using a standard curve.

[0037] Example 1 This example provides a screening method for ectoin-producing halophilic bacteria, including the following steps: (1) Add 1g of soil sample or 1mL of liquid sample to 9mL of high-salt culture medium, and culture at 30℃ and 200rpm for 3 days to obtain fermentation broth. Detect the ectoin content of the fermentation broth using HPLC to screen for ectoin-producing mixed bacterial groups; (2) Inoculate the ectoin-producing mixed bacterial group obtained in step (1) into high-salt culture medium, and culture at 30℃ and 200rpm for 7 days to obtain initial bacterial broth; Inoculate the initial bacterial broth into high-salt culture medium with a NaCl concentration of 4wt%, and culture at 30℃ and 200rpm for 7 days to obtain second-generation bacterial broth; Inoculate the second-generation bacterial broth into high-salt culture medium with a NaCl concentration of 4wt%. The third generation bacterial solution was obtained by culturing in a 5wt% high-salt medium at 30°C and 200rpm for 7 days. The third generation bacterial solution was inoculated into a 6wt% high-salt medium with NaCl concentration and cultured at 30°C and 200rpm for 7 days to obtain the fourth generation bacterial solution. The fourth generation bacterial solution was inoculated into a 7wt% high-salt medium with NaCl concentration and cultured at 30°C and 200rpm for 7 days to obtain the fifth generation bacterial solution. The fifth generation bacterial solution was inoculated into a 8wt% high-salt medium with NaCl concentration and cultured at 30°C and 200rpm for 7 days to obtain the sixth generation bacterial solution. The sixth generation bacterial solution is the enriched ectoin-producing halophilic bacterium. (3) The sixth generation bacterial solution obtained in step (2) was diluted 10 times in a gradient, and 10 times the diluted solution was taken. -4 10 -5 10 -6 and 10 -7 The bacterial suspensions were spread onto high-salt culture plates and incubated at 30°C for 7 days. Single colonies of different colors and shapes were picked and inoculated onto high-salt solid culture plates and incubated at 30°C for 7 days. Single colonies were picked and inoculated onto new high-salt solid culture plates and incubated at 30°C for 7 days. The culture was then passaged twice to obtain purified ectoin-producing halophilic bacteria. (4) The purified ectoin-producing halophilic bacteria obtained in step (3) were inoculated onto high-salt culture medium with a NaCl concentration of 7wt% and incubated at 37°C. After culturing at 200 rpm for 2 days, the ectoin content was detected by HPLC, and a halophilic bacterium with an ectoin content of 93±3.2 mg / L was screened and named GUYU39. (5) The GUYU39 obtained in step (4) was inoculated into a high-salt culture medium and cultured at 30℃ and 200 rpm for 7 days to obtain bacterial solution. The bacterial solution was mixed with 50v / v% glycerol at a volume ratio of bacterial solution: 50v / v% glycerol = 1:1 and stored in a -80℃ refrigerator.

[0038] Example 2 identifies GUYU39 obtained in Example 1. The specific method is as follows: 1. Observation of morphological characteristics.

[0039] GUYU39 was inoculated into agar plates No. 2216 and incubated at 28°C for 24 hours. The morphology of single colonies was observed and Gram staining was performed. Another single colony was negatively stained and observed using a transmission electron microscope. The results are shown in Figure 2.

[0040] As shown in Figure 2, the colonies of strain GUYU39 are all raised, translucent, and round with intact edges. GUYU39 are all aerobic, Gram-negative, non-spore-forming bacilli (0.9 × 2.0 μm), possessing peritrichous or lateral / polar flagella, and are motile.

[0041] 2. Molecular identification.

[0042]

[0043] The sequencing results were compared with the 16S rRNA gene of GUYU39 by BLAST on NCBI. The results showed that the similarity of the 16S rRNA gene of GUYU39 was 100%.

[0044] The 16S rRNA sequence of GUYU39 was uploaded to the EZBioCloud database for analysis and comparison. The results showed that GUYU39 is similar to the model bacterium Vreelandella massiliensis Marseille-P2426. T The highest similarity was observed (98.21%), followed by the model bacterium *Vreelandella sulfidaeris* ATCC BAA-803. T Based on the similarity (98.18%), GUYU39 is preliminarily identified as belonging to the genus Vreelandella.

[0045] Using Aidingimonas halophila DSM 19219T 11336 as the outer sequence, a phylogenetic tree was constructed using MEGA 7.0 software. The topological structure of the phylogenetic tree was evaluated using the Neighbor-Joining method with a Bootstrap value of 1000. The NJ tree is shown in Figure 3.

[0046] As shown in Figure 3, GUYU39 is most closely related to Vreelandella massiliensis Marseille-P2426 within the genus Vreelandella, and is preliminarily identified as a new species of Vreelandella. GUYU39 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36221, on October 9, 2025.

[0047] 3. To further determine the taxonomic position of GUYU39, whole-genome sequencing was performed on GUYU39.

[0048] Shanghai Meiji Biopharmaceutical Technology Co., Ltd. was commissioned to complete the extraction, quality control, and library construction of the bacterial strain's genomic DNA. Whole-genome sequencing of the target strain was performed using the Illumina Hiseq platform, yielding over 1GB of data, which was then assembled using SPADESV 3.15.0 software. Contigs shorter than 500bp and with coverage less than 10 were removed using Notepad++ software. The genome sequence was uploaded to the NCBI database to obtain accession numbers, and genome sequences of related type strains were also collected.

[0049] Using the ANI Calculator tool on the EzBioCloud platform, the genome sequences of the target strain and related type strains were uploaded, and their ANI values ​​were calculated. A 95%-96% threshold was used to determine whether different strains belonged to different species. The results showed that GUYU39 had an ANI value of 99.97%. Strain GUYU39 had an ANI value of 74.21% with the type strain Vreelandella massiliensis Marseille-P2426, which had the highest 16S rRNA gene similarity (98.21%), and an ANI value of 74.68% with the type strain Vreelandella sulfidaeris ATCC BAA-803, which had a similarity of 98.18%. All these values ​​were below the 95%-96% threshold for species identification.

[0050] Using the GGDC 3.0 tool on the German Society for the Preservation of Microbial Cultures (DSMZ) website, the genome sequences of the target strain and related type strains were uploaded, and the dDDH values ​​between the genomes of different strains were calculated. A 70% threshold was used to determine whether different strains belonged to different species. The results showed that GUYU39 had a dDDH value of 100%, while the dDDH value of the type strain Vreelandella massiliensis Marseille-P2426, which had the highest 16S rRNA gene similarity (98.21%), was 16%, less than the 70% threshold for species identification.

[0051] Therefore, GUYU39 was identified as a new species distinct from the type strain of the genus Vreelandella. Furthermore, the complete genome DNA of strain GUYU39 contains 3,683,220 base pairs, with a G+C content of 59.84%.

[0052] Example 3 tested the physiological and biochemical characteristics of GUYU39 obtained in Example 1. The specific method is as follows: 1. Salt tolerance.

[0053] GUYU39 cells were inoculated into LB medium and cultured at 37℃ and 200 rpm for 48 h. NaCl concentration gradients were set from 0 wt% to 20 wt% (0 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, 15 wt%, 18 wt%, 20 wt%, with three biological replicates for each concentration). OD values ​​were measured using a spectrophotometer. 600 The absorbance was measured in nm to determine the salt concentration range and optimal growth salt concentration of the strain. The results are shown in Table 1.

[0054] Table 1. Salt tolerance test results of GUYU39

[0055] As shown in Table 1, the salinity of GUYU39 ranges from 0wt% to 15wt%, indicating that it has good salt tolerance.

[0056] 2. Acid and alkali tolerance.

[0057] GUYU39 cells were inoculated into LB medium and cultured at 37℃ and 200 rpm for 48 h using a shaker. pH values ​​were set at 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. OD values ​​were measured using a spectrophotometer. 600nm The absorbance was used to determine the pH range and optimal growth pH of the strain, and the results are shown in Table 2.

[0058] Table 2 Results of acid and alkali tolerance test for GUYU39

[0059] As shown in Table 2, GUYU39 grows in a pH range of 6-8, with the optimal pH being 7.

[0060] 3. Ability to produce oxidase and catalase.

[0061] Contact enzyme: Take a filter paper disc, pick up a vigorous bacterial cell with an inoculation loop and spread it on the disc, then add 1% tetramethyl-p-phenylenediamine solution. A red color within 10 seconds indicates a positive result, a red color within 10-60 seconds indicates a delayed positive result, and a red color after 60 seconds indicates a negative result.

[0062] Oxidase: Take a glass slide, add a drop of 3% hydrogen peroxide, and use an inoculation loop to pick up vigorous bacterial cells and spread them in the hydrogen peroxide solution. If a large number of bubbles are produced, it is considered positive; if no bubbles are produced, it is considered negative.

[0063] The results showed that GUYU39 was positive for both catalase and oxidase, indicating that GUYU39 has the ability to produce catalase and oxidase.

[0064] 4. Enzyme activity.

[0065] The enzyme activity and other physiological and biochemical indicators of GUYU39 were tested according to the instructions of bioMérieux 25200 API ZYM enzyme activity test strips and 20050 API 20NE non-enteric Gram-negative bacillus identification kits. Reference bacteria were also tested under the same conditions.

[0066] The results of the API ZYM enzyme activity test strip showed that GUYU39 was positive for alkaline phosphatase, esterase, lipase, leucine aminopeptidase, and valine aminopeptidase, and weakly positive for lipase, cystine aminopeptidase, and naphthol-AS-Bl-phosphatase. All other reactions were negative.

[0067] The results of the API 20NE non-intestinal Gram-negative bacillus identification kit showed that GUYU39 was weakly positive for urease and β-glucosidase, and could grow using the carbon sources adipic acid and malic acid; all other reactions were negative.

[0068] 5. Carbon source utilization.

[0069] The identification was performed using a Biolog microbial identification system. The bacterial suspension was inoculated into a 96-well plate, and the absorbance changes of the two strains during their respiratory metabolism using different carbon sources were detected, as well as the turbidity differences caused by the growth of the microorganisms themselves. The BIOLOG identification system was used to read and analyze the utilization of various carbon sources by the strains.

[0070] The results showed that GUYU39 was insensitive to sodium lactate, rifamycin SV, lincomycin, vancomycin, and lithium chloride, but sensitive to fusobionic acid, D-serine, acetosine, dimethylaminetetracycline, guanidine hydrochloride, sodium tetradecanoate sulfate, tetrazolium violet, naphthylpyridinone acid, potassium tellurite, aztreonam, sodium bromate, and sodium butyrate. Tetrazolium blue showed a weak positive result. Carbon source utilization was positive for aminoacetyl-L-proline, L-alanine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, L-serine, L-lactic acid, L-malic acid, bromosuccinic acid, α-hydroxy-butyric acid, β-hydroxy-D,L-butyric acid, propionic acid, and acetic acid. Dextrin, gentiobiose, melibiose, α-D-glucose, D-fructose, D-galactose, 3-formylglucose, D-fructose, L-fructose, L-rhamnose, D-fructose-6-phosphate, D-aspartic acid, D-serine, D-galacturonic acid, L-galacturonic acid lactone, D-glucuronic acid, glucuronide, methyl pyruvate, D-methyl lactate, Tween 40, γ-amino-butyric acid, α-keto-butyric acid, and acetoacetic acid. Other carbon source utilization was negative.

[0071] 6. Analysis of chemical components (fatty acids, respiratory quinones, and polar lipids).

[0072] GUYU39 was inoculated into a high-salt culture medium to obtain bacterial culture. Fatty acid methyl esters were extracted, and the fatty acid composition was analyzed by gas chromatography. GUYU39 was freeze-dried, and respiratory quinones and polar lipids were extracted using organic reagents. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to analyze and identify the respiratory quinones, and two-dimensional thin-layer chromatography (TLC) was used to separate different polar lipids. Fatty acids with a content exceeding 5% were used as the main components. Specific gas chromatography results are shown in Table 3, respiratory quinone results in Table 4, and polar lipid results in Figure 4.

[0073] Table 3. Results of the detection of major fatty acids in GUYU39

[0074] As shown in Table 3, GUYU39 contains fatty acids whose main components are 3-hydroxylauric acid, a mixture of palmitoleic acid isomers, hexadecanoic acid, cycloheptadecanoic acid, octadecanoic acid monounsaturated fatty acids, and cyclononadecanoic acid.

[0075] Table 4. Detection results of respiratory quinones in GUYU39 (%) As shown in Table 4, the main respiratory quinones of GUYU39 are Q-9, with a small amount of Q-8.

[0076] As shown in Figure 4, GUYU39 contains polar lipids whose main components are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphatidylinositol mannose (PIM), and phospholipids (PL).

[0077] Example 4: To determine whether GUYU39 contains enzymes related to ectoin production, its whole genome was mined and analyzed. The specific procedure is as follows: Based on the KEGG database and Swissprot files, the sequenced genome was annotated and analyzed to find the protein sequences of genes related to phenolic acid degradation. The obtained protein sequences were analyzed and compared online using Uniprot (https: / / www.uniprot.org / blast / ). The correctness of the protein was ensured by verifying the functional protein sequences on the website, thereby determining that strain GUYU39 has genes related to ectoin synthesis.

[0078] Using the protein sequences of ectoin-related genes as reference sequences, 72 complete genomes downloaded from the LPSN website (https: / / lpsn.dsmz,de / genus / microbacterium) were analyzed using the BLAST tool in NCBI to study the genomic characteristics of the Microbacterium genus and its potential degradation associations. When the gene-protein sequence homology was >30%, it indicated that the genome contained gene sequences with phenolic acid degradation function.

[0079] Metabolic pathway information can be found using the KAAS automatic annotation server (https: / / www.genome.jp / kaas-bin / kaas_main); genome function can be analyzed through rapid annotation using the RAST subsystem (https: / / rast.nmpdr.org / rast.cgi); and gene function can be analyzed by uploading genes to the UniProt online server (www.uniprot.org / blast / ).

[0080] The results showed that GUYU39 contains a complete ectoine gene cluster. Overall, it has the highest similarity to the ectoine gene cluster of strain Halomonas sp. Bachu37, with the similarity of the three genes ranging from 84% to 88%.

[0081] ectA: The ectA gene showed the highest similarity (88%) to that of Halomonas sp. Bachu 37 (isolated from reed rhizosphere soil), and was RAST-annotated as 2-aminobutyric acid acetyltransferase (EC 2.3.1.178); ectB: The ectB gene showed the highest similarity (84%) to that of Halomonas sp. Bachu 37, and was RAST-annotated as 2-aminobutyric acid transaminase (EC 2.6.1.76); ectC: The ectC gene showed the highest similarity (87%) to that of Halomonas sp. Bachu 37, and was RAST-annotated as ectoin synthase (EC 4.2.1.108).

[0082]

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for screening ectoine-producing halophilic bacteria, characterized in that, Includes the following steps: S1. Add the collected samples to a high-salt culture medium and culture for 3-5 days to obtain a fermentation broth. Detect the ectoine content in the fermentation broth using high-performance liquid chromatography and screen for ectoine-producing mixed bacterial groups. S2. Inoculate the ectoine-producing mixed bacterial group obtained in step S1 into a high-salt culture medium with NaCl concentration increasing by 1 wt% and perform continuous subculturing at least 5 times to obtain enriched ectoine-producing halophilic bacteria. S3. Isolate and purify the enriched ectoine-producing halophilic bacteria obtained in step S2 to obtain purified ectoine-producing halophilic bacteria.

2. The screening method as described in claim 1, characterized in that, Includes at least one of the following (I) to (III): (I) In step S1, the high-salt culture medium is a culture medium containing at least 3 wt% NaCl; (II) In step S1, the culture conditions are 28-32℃, 180-220 rpm and 2-4 days; (III) In step S2, the culture conditions for continuous subculturing are 28-32℃, 180-220 rpm and 6-8 days.

3. A halophilic bacterium producing ectoine, characterized in that, The ectoin-producing halophilic bacterium is Vreelandella sp.; the 16S rRNA of the ectoin-producing halophilic bacterium has at least 90% nucleotide sequence identity with SEQ ID NO.1; the ectoin-producing halophilic bacterium expresses 2-aminobutyric acid acetyltransferase, 2-aminobutyric acid transaminase and ectoin synthase.

4. The ecdysin-producing halophilic bacterium as described in claim 3, characterized in that, The amino acid sequence of the 2-aminobutyric acid acetyltransferase is shown in SEQ ID NO.2, the amino acid sequence of the 2-aminobutyric acid transaminase is shown in SEQ ID NO.3, and the amino acid sequence of the ectoin synthase is shown in SEQ ID NO.

4.

5. A gene cluster for synthesizing ectoine, characterized in that, It includes ectA, ectB, and ectC; the nucleotide sequence of ectA is shown in SEQ ID NO.5, the nucleotide sequence of ectB is shown in SEQ ID NO.6, and the nucleotide sequence of ectC is shown in SEQ ID NO.

7.

6. A 2-aminobutyric acid acetyltransferase, characterized in that, The amino acid sequence of the 2-aminobutyric acid acetyltransferase is shown in SEQ ID NO.

2.

7. A 2-aminobutyric acid transaminase, characterized in that, The amino acid sequence of the 2-aminobutyric acid transaminase is shown in SEQ ID NO.

3.

8. An ectoin synthase, characterized in that, The amino acid sequence of the ectoin synthase is shown in SEQ ID NO.

4.

9. The use of the ectoin-producing halophilic bacteria as described in any one of claims 3-4, the gene cluster for synthesizing ectoin as described in claim 5, the 2-aminobutyric acid acetyltransferase as described in claim 6, the 2-aminobutyric acid transaminase as described in claim 7, or the ectoin synthase as described in claim 8 in the production of ectoin.

10. A method for producing ectoine, characterized in that, The process includes the following steps: inoculating the ectoin-producing halophilic bacteria according to any one of claims 3-4 into a high-salt culture medium for fermentation to obtain a fermentation broth, and purifying the fermentation broth to obtain ectoin.

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