A strain of Gymnodinialimonas sp.57CJ19 and its application

Abstract

The present invention belongs to the field of microorganism and ecological restoration technology, and specifically relates to a strain ( Gymnodinialimon ) sp.57CJ19 bacteria and their applications. The bacteria are deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No. 30521. The above bacterial strain was inoculated in PHBA stress medium. After cultivation, the results showed that the strain could grow using PHBA. The strain had obvious reproduction within less than 10 hours of inoculation, and the PHBA concentration decreased rapidly. After about 30 hours of cultivation, the PHBA concentration dropped to half of the original concentration, proving that G. sp. 57CJ19 has excellent degradation effect on PHBA.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and ecological restoration, and specifically relates to a strain of marine-derived (Gymnodinialimonas) sp.57CJ19 bacteria and an application thereof. Background Art

[0002] Para-hydroxybenzoic acid (PHBA) is an aromatic compound widely found in nature and an intermediate metabolite of many natural products and synthetic compounds. PHBA and its salts are widely used as preservatives in daily chemicals and agricultural chemicals. PHBA may also be used or produced in the production process of some chemical products and medicines. PHBA and its derivatives are discharged into the environment and even into the ocean through domestic sewage and industrial wastewater, causing pollution of terrestrial soil, water environment and marine water bodies, posing potential risks to plants, animals and humans. Certain plants such as cotton, strawberries, cucumbers, tomatoes, tobacco, etc. naturally produce PHBA during metabolism, which enters the soil environment through natural decomposition processes, not only polluting the environment, but also producing self-toxicity, causing crop continuous cropping problems, affecting crop growth and development, reducing crop yield and quality, and damaging crop economic benefits.

[0003] At present, chemical degradation and bioremediation are the main methods for treating PHBA pollution. The degradation principle of PHBA aromatic pollutants is that microorganisms can convert PHBA into protocatechuic acid through the action of monooxygenase, and finally convert it into acetyl-CoA and succinyl-CoA to enter the citric acid cycle under the action of a series of hydrolases, hydratases and aldolases. The catechol ring cleavage products formed during the degradation of PHBA will inhibit the utilization of PHBA, which is manifested in different strains of PHBA degradation ability and substrate tolerance. Aromatic compounds such as PHBA are widely present in the environment. It is of great significance to explore new PHBA-degrading bacteria, obtain pure cultures to verify their degradation ability and substrate tolerance, and explain their degradation pathways, key enzymes and intermediates based on omics analysis, which is of great significance to enrich the bacterial resources related to pollutant degradation and the subsequent development of new and efficient degradation agents.

[0004] In 2022, Jiang et al. isolated a strain of bacteria from a rare marine dinoflagellate and determined its genome sequence. They believed that the bacteria was a new genus and representative species of the Rhodobacteraceae family and named it Gymnodinialimonas ceratoperidiniiJ12C1-MA-4. T (G.ceratoperidinii J12C1-MA-4 T)(Arch Microbiol 204(3):185(2022))). In 2023, Peng et al. isolated a strain from the highly toxic marine dinoflagellate Gymnodinialimonas phycosphaerae and determined its genome sequence, naming it Gymnodinialimonas phycosphaerae N5. T (G. phycosphaerae N5 T )(Int J Syst Evol Microbiol 73:005874(2023)). Currently, the genus Gymnodinialimonas only includes the above two species in the database, both of which were isolated from seaweed microbial communities; there is currently little research on this genus, and its metabolic and genomic characteristics are still unclear. Summary of the invention

[0005] The invention provides a marine-derived (Gymnodinialimonas) sp.57CJ19 bacterium and application of the bacterium in degrading aromatic compounds, especially in degrading PHBA.

[0006] The strain screened and protected by the present invention is (Gymnodinialimonas) sp.57CJ19 bacteria, abbreviated as G.sp.57CJ19, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 7, 2024, with the deposit number CGMCC No.30521.

[0007] The G.sp.57CJ19 bacterium provided by the present invention is a new species of the genus Gymnodinialimonas, and has unique physiological and biochemical characteristics. For example, the bacterium can degrade aromatic compounds, especially PHBA, and has excellent degradation effect. Therefore, the use of G.sp.57CJ19 in degrading aromatic compounds is the key content to be protected by the present invention; similarly, the present invention also focuses on protecting the use of G.sp.57CJ19 in degrading PHBA.

[0008] Taking PHBA as an example, the method of degrading it using G.sp.57CJ19 is as follows:

[0009] G.sp.57CJ19 was inoculated into PHBA stress medium and cultured at 200rpm and 20℃. The results showed that the strain could utilize PHBA for growth. The strain had obvious reproduction within 10h after inoculation, and the PHBA concentration decreased rapidly. After about 30h of culture, the PHBA concentration was reduced to half of the original concentration. This shows that the strain has a high utilization rate of PHBA, proving that G.sp.57CJ19 has excellent PHBA degradation effect. Regarding the ability of G.sp.57CJ19 to efficiently degrade aromatic compounds, Example 6 of the present invention provides a detailed analysis of the degradation pathway.

[0010] The present invention also protects a bacterial agent containing any one of the G.sp.57CJ19 bacteria, its fermentation product or its metabolites;

[0011] The present invention particularly protects the bacterial agent obtained by fermentation culture of G.sp.57CJ19 bacteria;

[0012] The above-mentioned bacterial agent is any one of a liquid bacterial agent, a powdered bacterial agent or a granular bacterial agent. Of course, the above are only examples, and other forms of bacterial agents are also possible. Changes to the appearance of the bacterial agent of the present invention also fall within the protection scope of the present invention.

[0013] The beneficial effects of the present invention are:

[0014] The present invention provides a novel marine-derived bacterium G.sp.57CJ19, which has an excellent degradation effect on aromatic compounds, especially PHBA. Specifically, 30 hours after G.sp.57CJ19 is inoculated in a PHBA stress medium, the PHBA concentration is reduced to half of the original concentration.

[0015] If G.sp.57CJ19 is applied to the degradation of aromatic compounds, it will provide a new way for the safe and efficient degradation of such pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 In Example 1 of the present invention, the colony morphology of G.sp.57CJ19 was visually observed;

[0017] Figure 2 The morphology (A) and Gram staining result (B) of G.sp.57CJ19 under oil immersion microscope in Example 1 of the present invention;

[0018] Figure 3 Phylogenetic tree of G.sp.57CJ19 in Example 2 of the present invention; the numbers on the branches represent the bootstrap values, and the larger the bootstrap value, the higher the confidence of the node;

[0019] Figure 4Degradation of PHBA by G.sp.57CJ19 in Example 5 of the present invention;

[0020] Figure 5 Degradation pathway analysis (A) and gene cluster analysis (B) of PHBA degradation by G.sp.57CJ19 in Example 6 of the present invention. DETAILED DESCRIPTION

[0021] (Gymnodinialimonas) sp.57CJ19 refers to the bacteria mentioned in the content of the invention, referred to as "57CJ19" or "G.sp.57CJ19" in the following examples).

[0022] Example 1 Isolation and morphological observation of 57CJ19

[0023] Chitin liquid medium: 0.5% powdered chitin, 0.01% yeast powder, 0.01% ammonium chloride, 0.1% sodium nitrate, 0.1% vitamin solution, 1.5% agar, fresh seawater, pH 7.5.

[0024] Chitin solid medium: 0.05% N-acetylglucosamine, 0.05% peptone, 0.01% yeast powder, 0.1% vitamin solution, 1.5% agar, fresh seawater, pH 7.5.

[0025] Zobell 2216E medium: 5 g peptone, 1 g yeast extract, 0.01 g ferric phosphate, 15 g agar, 1000 mL Chenhai water, pH 7.5 adjusted with NaOH.

[0026] Take 2g of surface sediment sample from the intertidal zone of Aoshan Bay, Qingdao, and culture it in 100mL chitin liquid culture medium at 20℃ and 200rpm for 2-100d. -5 and 10 -6 Dilution degree, take 100 μL of culture to spread on chitin solid culture medium plate, put the plate upside down in 20 ℃ incubator and culture for a certain period of time, select the colonies with good growth on the plate and streak on the chitin solid plate for 3 times to purify the strains, and eliminate the strains with weak growth ability during the subculture process.

[0027] A strain was obtained by screening through the above method and named 57CJ19. The colonies of this strain on 2216E solid medium were white, round, and about 1-1.5 mm in diameter ( Figure 1 ). The oil microscope magnified 600 times and observed that the strain was short rod-shaped, about 2-3 μm long and about 0.5-1 μm wide ( Figure 2 A); Gram staining method observed that the cell wall of the strain was red after staining, which was Gram-negative bacteria ( Figure 2 B).

[0028] Example 2 Identification of 57CJ19 strain

[0029] Take 1 mL of 57CJ19 bacterial solution and extract total bacterial DNA according to the instructions of the "Bacterial Genome Extraction Kit" (DP2001, Bioteke).

[0030] The 16S universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3') were designed. The bacterial genome was used as a template, and the DNA fragments were obtained by PCR according to the PCR system and procedure in the instruction manual of Taq Plus Master Mix II (P213, Vazyme). The DNA fragments were recovered using a DNA gel recovery kit (DC301, Vazyme). The recovered PCR products were sequenced at Beijing Qingke Biotechnology Co., Ltd.

[0031] The total length of the 16S rRNA sequence measured by sequencing was 1388bp. The sequence was compared and analyzed in the EzBioCloud database (https: / / www.ezbiocloud.net / ). The results showed that strain 57CJ19 was in the same branch as the Maribius genera and Jannaschia genera of the Rhodobacteraceae and was in a parallel relationship. The strain was named Jannaschia sp. strain 57CJ19. The 16S rRNA sequence of strain 57CJ19 was submitted to the NCBI Genbank database in June 2022, with GenBank number OR078505.1, and the sequence was made public in the database in June 2023.

[0032] In 2022, Jiang et al. isolated a strain of bacteria from a rare marine dinoflagellate and determined its genome sequence. They believed that the bacteria was a new genus and representative species of the Rhodobacteraceae family and named it Gymnodinialimonas ceratoperidiniiJ12C1-MA-4. T (G.ceratoperidinii J12C1-MA-4 T )(Arch Microbiol 204(3):185(2022))). In 2023, Peng et al. isolated a strain from the highly toxic marine dinoflagellate Gymnodinialimonas phycosphaerae and determined its genome sequence, naming it Gymnodinialimonas phycosphaerae N5. T (G. phycosphaerae N5 T)(Int J Syst Evol Microbiol 73:005874(2023)). Currently, the genus Gymnodinialimonas only includes the above two species in the database, both of which were isolated from seaweed microbial communities; there is currently little research on this genus, and its metabolic and genomic characteristics are still unclear.

[0033] Since the bacteria of the genus Gymnodinialimonas were first discovered and named in 2022, the 16SrRNA sequence of strain 57CJ19 was compared and analyzed in the EzBioCloud database, showing that it is similar to G.ceratoperidinii J12C1-MA-4 T A segment of the whole genome sequence has a sequence identity of 97.91%. It is generally recognized that 16S rRNA sequences have been widely used in the classification and identification of bacteria and archaea. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish the taxonomic relationship between prokaryotic strains. It is currently believed that 98.65% similarity is the cutoff value for species division, that is, 16S rRNA similarity lower than 98.65% can be identified as a new species (Microbiol Today 4(4):6-9(2006); Method Microbiol 41:61-74(2014)). Therefore, G.sp.57CJ19 belongs to a new species of this genus. By constructing a phylogenetic tree, it is shown that strain 57CJ19 is similar to G.ceratoperidinii J12C1-MA-4 T and G. phycosphaerae N5 T The relationship of belonging to the same branch and being parallel further confirmed that strain 57CJ19 is a new species of the genus Gymnodinialimonas ( Figure 3 ).

[0034] After the complete genome sequence of 57CJ19 bacteria was subsequently determined, it was named Gymnodinialimonassp.57CJ19 (G.sp.57CJ19).

[0035] Example 3 G.sp.57CJ19 genome sequencing

[0036] Take 200mL of 57CJ19 bacterial solution, centrifuge at 4℃, 8000r / min for 10min, resuspend in 1x PBS, centrifuge at 4℃, 8000r / min for 10min, repeat twice. The bacterial sludge was sent to Shanghai Paisono Biotechnology Co., Ltd. for sequencing. The total bacterial DNA was extracted using the CTAB method, and the genome sequencing platform was Illumina NovaSeq PE150 and Nanopore sequencing technology.

[0037] The clean data of the second-generation sequencing was spliced ​​using the software SOAPdenovo, the spliced ​​scaffold was filled with holes using the software GapCloser, the genome quality was assessed using the software Checkm (parameter: lineage_wf), and the repetitive sequences of the genome were removed using the software RepeatMasker. Second-generation sequencing data quality control Third-generation single-molecule sequencing data assembly: The third-generation data was assembled using the software Unicycler and Flye to obtain the contig sequence. The second-generation data corrected the third-generation results: The second-generation high-quality data was corrected using the software pilon to correct the third-generation contig results, and finally the complete sequence was spliced. RNA was predicted using the software RNAmmer (parameter: -multi), and the software Prodigal was used to predict genes and output the corresponding amino acid sequence files.

[0038] We selected the whole genome sequences of 10 species with close similarity to the 16S rRNA sequence of G.sp.57CJ19, and calculated the average nucleotide identity (ANI) between the genome of each species and its close relatives; we used the Genome-to-Genome Distance Calculator (GGDC v 3.0, http: / / ggdc.dsmz.de / distcalc2.php) to calculate the digital DNA:DNA hybridization (dDDH) between the genome of each species and its close relatives. The specific results are shown in Table 1.

[0039] The maximum average nucleotide identity of G.sp.57CJ19 with closely related species is 79.87%, and the maximum dDDH value is only 22.2%. It is generally recognized that when the ANI value of a species with its closely related strains is less than 95%-96%, it can be classified as a potential new species (Proc Natl Acad Sci US A106(45):19126-19131(2009)); when the dDDH value is 20%-70%, it is inferred that the strain is a different species within the same genus (Int J Syst Evol Microbiol57(Pt 1):81-91(2007)). Based on the classification results of ANI and dDDH, the strain G.sp.57CJ19 was confirmed to be a new species of the genus Gymnodinialimonas.

[0040] The whole genome data of G.sp.57CJ19 was submitted to the NCBI Genbank database, and the Genbank number is CP151587.1.

[0041] Table 1 ANI and dDDH of G.sp.57CJ19 homologous strains

[0042]

[0043]

[0044] Example 4 Physiological and biochemical assays of G.sp.57CJ19

[0045] 1. Nitrate reduction capacity

[0046] Add the bacterial solution to the nitrate reduction medium at a 2% inoculum volume, culture at 30°C, 200rpm until turbid, take 1mL of the bacterial solution in a centrifuge tube, mix 100μL of each of the A and B solutions in the kit, and add 100μL of the mixed solution to the bacterial solution. If the bacterial solution turns red, it has nitrate reduction ability (positive); if the bacterial solution does not turn red, add an appropriate amount of zinc powder. If the bacterial solution turns red, it does not have nitrate reduction ability (negative), and if it does not turn red, it is a false positive.

[0047] The results showed that G.sp.57CJ19 was unable to reduce nitrate.

[0048] 2. Starch hydrolysis ability

[0049] Streak the bacterial liquid onto starch hydrolysis medium, and add Lugol's iodine solution after colonies grow. If a transparent circle is formed around the colonies, it has the ability to hydrolyze starch.

[0050] The results showed that G.sp.57CJ19 had the ability to hydrolyze starch.

[0051] 3. Ability to utilize the sole carbon source

[0052] The bacterial liquid was added to the sole carbon source utilization medium at a 2% inoculum, and the medium was cultured at 30°C and 200rpm for 2 days to observe whether it was turbid. If it was turbid, it proved that the strain could utilize the carbon source. Sugars or sugar metabolites used as the sole carbon source include: trehalose, D-mannitol, D-mannose, D-arabinose, sucrose, D-xylose, D-galactose, D-fructose, D-ribose, maltose, acetate, cellobiose, glucose, raffinose, citrate and malate.

[0053] The results showed that the strain could utilize mannitol, D-mannose, sucrose, D-xylose, D-galactose, D-fructose, D-ribose, maltose, cellobiose, glucose, and raffinose, but could not utilize trehalose, D-arabinose, citrate, acetate, and malate.

[0054] The phenotypic results of G.sp.57CJ19 related strains published in the BacDive database (https: / / bacdive.dsmz.de / ) and the IJSEM journal were compared with the phenotypic results of G.sp.57CJ19 disclosed in the present invention, and the comparison results with significant differences are listed in Table 2. For example, (1) 57CJ19 can utilize D-mannitol and D-mannose, while J12C1-MA-4 of the same genus can T cannot utilize it; (2) 57CJ19 can utilize sucrose and raffinose, while strains of the closely related genus Jannaschia cannot utilize them; 57CJ19 can utilize cellobiose, while most strains of the closely related genus Jannaschia cannot utilize it; (3) 57CJ19 cannot utilize arabinose, while strains of the closely related genus Jannaschia can utilize it.

[0055] The above reveals that the G.sp.57CJ19 provided by the present invention has unique physiological and biochemical characteristics and is novel.

[0056] Table 2 Physiological and biochemical comparison of G.sp.57CJ19 and its related strains

[0057]

[0058] Note: "+" indicates positive result; "-" indicates negative result; "w" indicates weak positive result; nd: undetermined. Except for strain 57CJ19, the phenotypic results of closely related species were derived from IJSEM literature records and BacDive database.

[0059] 4.API zym enzyme activity test

[0060] The API zym results of G.sp.57CJ19 are shown in Table 3. Strain 57CJ19 has no acid phosphatase activity, which is completely different from the closely related strains; strain 57CJ19 has no alkaline phosphatase activity, which is completely different from the vast majority of closely related strains; strain 57JC19 has α-glucosidase activity, and the closely related strains have almost no enzyme activity (Table 4). The above reveals that the G.sp.57CJ19 provided by the present invention has unique physiological and biochemical characteristics and is novel.

[0061] Table 3 G.sp.57CJ19 API zym test results

[0062]

[0063] Note: “+” represents positive, with corresponding enzyme activity; “-” represents negative, with no corresponding enzyme activity.

[0064] Table 4 Comparison of enzyme activities between G.sp.57CJ19 and related strains (partial)

[0065]

[0066]

[0067] Example 5 G.sp.57CJ19 Degradation of PHBA

[0068] Zobell 2216E medium: 5 g peptone, 1 g yeast extract, 0.01 g ferric phosphate, 15 g agar, 1000 mL Chenhai water, pH 7.5 adjusted with NaOH.

[0069] 2216E liquid culture medium: 5 g peptone, 1 g yeast extract, 1000 mL fresh seawater, pH adjusted to 7.0.

[0070] PHBA was prepared into a 1.0 g / L mother solution with acetone, and then sterilized by filtration through a 0.22 μm organic filter membrane for later use. PHBA was added to 2216E solid medium and liquid medium at a concentration of 100 μg / mL and 500 μg / mL, and the pH was adjusted to 7.0 with NaOH.

[0071] 100 μL of the G.sp.57CJ19 culture was applied to a 2216E solid medium plate supplemented with PHBA, and the plate was inverted and placed in a 20°C incubator for 7 days, and colonies were observed growing on the plate. The above indicates that G.sp.57CJ19 can tolerate PHBA and utilize PHBA for growth.

[0072] G.sp.57CJ19 bacterial suspension was inoculated into 2216E liquid medium containing 100 μg / mL and 500 μg / mL PHBA at an inoculum size of 2%, respectively, and cultured at 200 rpm and 20°C for 1 day. The medium was observed to be obviously turbid. The above shows that G.sp.57CJ19 can tolerate a higher concentration of PHBA for growth.

[0073] PHBA stress medium: 0.25% D-glucose, 0.25% D-mannose, 0.25% maltose, 0.25% cellulose, 0.15% NH 4 Cl, 0.02% casein acid hydrolyzate, 100 μg / mL PHBA, fresh seawater, pH adjusted to 7.0 with NaOH.

[0074] The strain 57CJ19 was inoculated into the PHBA stress medium and cultured at 200 rpm and 20°C. The concentration of PHBA in the culture was determined by measuring the UV absorbance at 273 nm, and the growth of bacteria was determined by measuring the absorbance at 600 nm. When determining the PHBA concentration, the culture medium without PHBA was used as a control. The specific results are shown in Figure 4 , Table 5 and Table 6 ( Figure 4 The tables corresponding to the detected data are shown in Table 5 and Table 6). Figure 4 In the Figure 1, the concentration of PHBA in the culture was determined by measuring the UV absorbance at 273 nm (blue dots), and the growth of bacteria was determined by measuring the absorbance at 600 nm (red squares).

[0075] Table 5 OD of strain 57CJ19 600 Table of value changes over time

[0076]

[0077] Table 6 PHBA concentration (μg / mL) changes with culture time

[0078]

[0079] The results showed that the strain could utilize PHBA for growth. The strain reproduced significantly within 10 hours after inoculation. Meanwhile, the PHBA concentration decreased rapidly. After about 30 hours of culture, the PHBA concentration dropped to half of the original concentration, indicating that the strain had a high utilization rate of PHBA.

[0080] The above is just an example of PHBA to prove that G.sp.57CJ19 has excellent degradation effect on aromatic compounds. The degradation of G.sp.57CJ19 on such substances is not limited to PHBA. Example 6 will further prove that G.sp.57CJ19 has excellent degradation effect on aromatic compounds and other pollutants through degradation pathway analysis.

[0081] Example 6 Analysis of PHBA degradation pathways of G.sp.57CJ19

[0082] The genome obtained in Example 3 was annotated for gene function using eggNOG. The results showed that the G.sp.57CJ19 genome encodes a complete PHBA metabolic pathway. G.sp.57CJ19 has a gene encoding 4-hydroxybenzoate 3-monooxygenase (pobA, EC 1.14.13.2), which can catalyze the conversion of PHBA to 3,4-dihydroxybenzoate (protocatechuate). G.sp.57CJ19 also has a full set of genes for converting 3,4-dihydroxybenzoate (protocatechuate) to succinyl-CoA and acetyl-CoA through the protocatechuate branch of the β-ketoadipate pathway. The enzymes encoded by these genes include protocatechuate 3,4-dioxygenase (pcaG, pcaH, EC 1.13.11.3), 3-carboxy-cis, cis-xylindole cycloisomerase (pcaB, EC5.5.1.2), 4-carboxy-xylindole lactone decarboxylase (pcaC, EC 4.1.1.44), 3-oxoadipate enol lactonase (pcaD, EC3.1.1.24), 3-oxoadipate-CoA transferase (pcaI, pcaJ, EC 2.8.3.6), 3-oxoadipate-CoA sulfhydrylase (pcaF, EC 2.3.1.174), and acetyl-CoA acyltransferase (fadA, EC 2.3.1.16), see Figure 5 A. The above genes are distributed in several gene clusters, with two pcaC genes and two pcaD genes, see Figure 5 B, Figure 5 The right arrow in B indicates that the gene is on the forward strand, the left arrow indicates that the gene is on the reverse strand, and the number inside the arrow indicates the nucleotide length of the gene. This is also an important way for bacteria to degrade phthalic acid and microorganisms to degrade other aromatic compounds. Therefore, G.sp.57CJ19 can not only degrade PHBA, but also degrade pollutants such as aromatic compounds.

Claims

1. A strain of bacteria Gymnodinialimonas sp.57CJ19, characterized in that The bacteria are deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No. 30521.

2. A bacterium as claimed in claim 1 Gymnodinialimonas Application of sp.57CJ19 in degradation of p-hydroxybenzoic acid.

3. A bacterial agent, characterized in that: The bacterial agent contains the bacteria as claimed in claim 1 Gymnodinialimonas sp.57CJ19.

4. A bacterial agent as claimed in claim 3, characterized in that: The bacterial agent is composed of bacteria Gymnodinialimonas sp . Obtained by fermentation culture of 57CJ19.

5. A bacterial agent as claimed in claim 4, characterized in that: The bacterial agent is any one of a liquid bacterial agent, a powdery bacterial agent or a granular bacterial agent.

6. Use of the bacterial agent according to any one of claims 3 to 5 in degrading p-hydroxybenzoic acid.

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