Marinomonas and its application

Abstract

The present invention provides a Marinomonas sp. and its application. Specifically, the present invention unexpectedly finds a new strain named Marinomonas algarum from red algae. The Marinomonas algarum of the present invention has the function of producing ectoine. The present invention also discloses a new Marinomonas sp. strain E8.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to Marinomonas sp. separated from red alga Gelidium amansii and applications thereof. Background Art

[0002] The genus Marinomonas belongs to the family Marine Spirochaetaceae of the phylum Gammaproteobacteria and was created by Van Landschoot and De Ley (1984) to accommodate two reclassified species, Marinomonas vaga and Marinomonas communis. As of April 2022, the genus has 39 published species isolated from marine environments (https: / / lpsn.dsmz.de / genus / marinomonas), of which 11 are from marine plants, 10 from seawater, and 5 from marine sediments. Many of them have been observed to have special properties, such as agar degradation (M. agarivorans KCTC 52475T), algicidal activity (M. algicida KEMB 9005-327 T ), beneficial properties of plants (M.spartinae KCTC 42958 T ) and thermophilic characteristics (M.primoryensis JCM 11775 T ). All strains of the genus Marinomonas are Gram-negative, rod-shaped, strictly aerobic, and some of them can hydrolyze agar and gelatin, and have the ability to metabolize quinic acid, acetic acid, glycerol, and lactic acid. Studies have found that the genus Marinomonas exhibits strong adaptability to special marine environments. Different secondary metabolites have been detected in some strains of the genus Marinomonas, including Marinomonas fungiae JCM18476 T Marinomonas mediterranea MMB-1 can produce ω-ester-containing peptides (OEPs) and marinocine, which has certain antibacterial potential. Summary of the invention

[0003] The purpose of the present invention is to provide a new species of the genus Marinomonas. According to morphological, physiological and biochemical characteristics and chemical taxonomic characteristics, the strain E8 of Marinomonas is a new species of the genus Marinomonas. Comparative genome analysis also shows that the strain E8 of Marinomonas is a new species of the genus Marinomonas, so the new species is named Marinomonas algarum. The new strain E8 is subjected to multiphase classification analysis by a multiphase classification method. The pan-genome of the bacterium and the homologous proteins of the genus are preliminarily analyzed, and the biosynthetic gene clusters related to secondary metabolism are predicted.

[0004] A first aspect of the present invention provides a Marinomonas, wherein the Marinomonas is Marinomonas Marinomonasalgarum.

[0005] In another preferred example, the sequence of the 16s rRNA of the Seamonas is as shown in SEQ ID NO.1; or the sequence of the 16s rRNA of the Seamonas has ≥99% homology compared with the sequence shown in SEQ ID NO.1, preferably, has ≥99.5% homology, and more preferably has ≥99.8% homology (for example, has ≥99.9% homology).

[0006] In another preferred embodiment, the Marinomonas is Marinomonas sp. E8 strain, and its preservation number is CGMCC NO.25012.

[0007] In another preferred embodiment, the Marinomonas sp. is a derivative strain of the Marinomonas sp. E8 strain, such as a strain obtained by gene mutation or genetic engineering.

[0008] In another preferred example, the Marinomonas is from algae.

[0009] In another preferred embodiment, the Marinomonas is from red algae.

[0010] In another preferred embodiment, the Marinomonas is from the red alga Gelidium amansii.

[0011] The second aspect of the present invention provides a microbial agent, which includes: the Marinomonas and / or its metabolites described in the first aspect of the present invention.

[0012] In another preferred embodiment, in the microbial agent, the content of Marinhomonas and / or its metabolites is 1×10-1×10 15 cfu / mL or cfu / g Marinomonas algarum, preferably 1×104 -1×10 9 cfu / mL or cfu / g Marinomonas algarum, based on the total volume or weight of the inoculum.

[0013] In another preferred embodiment, the bacterial agent contains 0.0001-99 wt%, preferably 0.1-90 wt% of the Marinomonas and / or its metabolites, based on the total weight of the bacterial agent.

[0014] In another preferred embodiment, the dosage form of the bacterial agent is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.

[0015] The third aspect of the present invention provides a use of the Marinomonas described in the first aspect of the present invention or the bacterial agent described in the second aspect of the present invention for fermentation to produce ectoine or for wastewater treatment.

[0016] In another preferred embodiment, the wastewater is high-salt wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown are transmission electron micrographs of E8 cells. Cells were cultured in 2216E medium at 28°C for 48 hours. Scale bar, 500 nm.

[0018] Figure 2 The maximum likelihood phylogenetic tree based on the 16S rRNA gene sequences of strain E8 from other members of the Oceanospirillaceae is shown. All strains of the genus Marinomonas have been identified. The branch nodes (ML / NJ / MP) show the bootstrap values ​​(greater than 0%) of 1000 replicates. T (GenBank accession number MG706141) is the outgroup. Bar, 0.01 substitution at each nucleotide position.

[0019] Figure 3 The phylogenomic tree reconstructed by IQtree shows the position of strain E8 and related strains of Oceanospirillaceae and the reference genome sequence. GenBank accession numbers are in brackets. Bootstrap values ​​(expressed as percentage of 1000 replicates) exceeding 70% are shown at branch points. Zobellella endophytica 59N8T (GenBank accession number PXYG00000000) was used as outgroup. Bar, 0.2 substitutions per nucleotide position.

[0020] Figure 4Two-dimensional TLC plates of strain E8 and control strains after lipid extraction are shown. The plates were sprayed with 5% (v / v) phosphomolybdic acid to show the presence of all lipids. A, strain E8; B, Marinomonas pontica DSM 17793 T ;C, Marinomonas communis JCM 20766 T .

[0021] Figure 5 A heat map showing the pairwise distances between a pair of genomes. Strains: 1, M. algarum E8; 2, M. agarivorans QM202 T ; 3, M. alcarazii CECT 7730 T ; 4, M.algicola SM1966 T ; 5, M. aquimarina CECT 5080 T ;6,M.aquiplantarum CECT 7732 T ;7,M.arctica BSI20414;8,M.atlantica Cmf 18.22 T ;9,M.balearica CECT 7378 T ; 10, M. colpomeniae SM2066 T ; 11, M. communis DSM 5604 T ; 12, M.flavescens ANRC-JHZ47 T ;13,M.foliarum JZW;14,M.fungiae JCM 18476 T ;15,M.gallaica CECT 5116 T ; 16, M. hwangdonensis HDW-15 T ; 17, M. mediterranea MMB-1 T ;18,M.ostreistagni UST010306-043 T ; 19, M. piezotolerans YLB-05 T ; 20, M. polaris DSM 16579 T ;21,M.pollencensis CECT 7375 T ;22,M.posidonica IVIA-Po-181 T;23,M.primoryensis MPKMM3633 T ; 24, M. profundi M1K-6 T ; 25, M.profundimarisD104 T ; 26, M. rhizomae IVIA-Po-145 T ; 27, M. shanghaiensis DSL-35 T ; 28, M. spartinae CECT 8886 T ; 29, M. ushuaiensis DSM 15871 T ; 30, M. vulgaris A79 T .

[0022] Figure 6 The average nucleotide identity (ANI) and average amino acid identity (AAI) of the strain E8 genome sequence are shown in pairwise comparisons with the Marinomonas reference genome sequence. Upper triangle: ANI value; lower triangle: AAI value.

[0023] Figure 7 The single-copy core protein tree shows the branches of the 30 strains.

[0024] Figure 8 The core pangenogram of the Marinomonas genus reference genome is shown.

[0025] Fig. 9 The pan-genome phylogenetic tree generated by the BPGA pipeline is shown.

[0026] Fig.10 A phylogenetic tree of the core genome generated by the BPGA pipeline is shown.

[0027] Fig.11 The KEGG distribution of core genes, essential genes, and unique genes of the genus Marinhomonas is shown.

[0028] Fig.12 A venn diagram constructed by the OrthoVenn2 online service is shown, showing the distribution of shared gene families (orthologous clusters) in strain E8 and four marine plant-related strains.

[0029] Fig.13The relative abundance of homologous protein groups for each strain is shown to function in it. RNA processing and modification are not annotated. E, amino acid transport and metabolism; R, general function prediction only; G, carbohydrate transport and metabolism; T, signal transduction mechanism; K, transcription; J, translation, ribosome structure and biogenesis; Z, cytoskeleton; W, extracellular structure; X, Mobilome: prophage, transposon; D, cell cycle control, cell division, chromosome segmentation; U, intracellular transport, secretion and vesicular transport; F, nucleotide transport and metabolism; V, defense mechanism; N, cell activity; Q, secondary metabolite biosynthesis, transport and degradation; O, post-translational modification, protein turnover, chaperone; I, lipid transport and metabolism; S, unknown function; L, replication, recombination and repair; M, cell wall / membrane / envelope biogenesis; C, energy production and conversion; P, inorganic ion transport and metabolism; H, coenzyme transport and metabolism; strains: 1, M.algarum E8; 2, M.agarivorans QM202 T ; 3, M. alcarazii CECT 7730 T ; 4, M.algicola SM1966 T ; 5, M. aquimarina CECT 5080 T ;6,M.aquiplantarum CECT 7732 T ;7,M.arctica BSI20414 T ; 8, M. atlantica Cmf 18.22 T ;9,M.balearica CECT 7378 T ; 10, M. colpomeniae SM2066 T ; 11, M. communis DSM 5604 T ; 12, M.flavescens ANRC-JHZ47 T ; 13, M.foliarum JZW T ; 14, M. fungiae JCM 18476 T ;15,M.gallaica CECT 5116 T ; 16, M. hwangdonensis HDW-15 T ; 17, M. mediterranea MMB-1 T ;18,M.ostreistagni UST010306-043 T ; 19, M. piezotolerans YLB-05 T; 20, M. polaris DSM 16579 T ;21,M.pollencensis CECT7375 T ;22,M.posidonica IVIA-Po-181 T ;23,M.primoryensis MPKMM3633 T ; 24, M.profundiM1K-6 T ; 25, M. profundimaris D104 T ; 26, M. rhizomae IVIA-Po-145 T ; 27, M. shanghaiensis DSL-35 T ; 28, M. spartinae CECT 8886 T ; 29, M. ushuaiensis DSM 15871 T ; 30, M. vulgaris A79 T .

[0030] Fig.14 Shown is the discovery of biosynthetic gene clusters in the genomes of strain E8 and Marinomonas reference strains using antiSMASH 6.0 software. DETAILED DESCRIPTION

[0031] After extensive and in-depth research and experiments, the inventors unexpectedly discovered a new strain named Marinomonas algarum from algae (such as red algae). The fermentation metabolites of the Marinomonas E8 strain of the present invention contain high levels of ectoine, and thus can be used to ferment and produce ectoine. On this basis, the present invention was completed.

[0032] As used herein, the terms "strain of the present invention", "Marinomonas algarum of the present invention", "Marinomonas sp. E8", "Marinomonas algarum", "Marinomonas of the present invention", and "Marinomonas" are used interchangeably and refer to the strain with the accession number: CGMCC NO.25012 and its successive strains or derivative strains.

[0033] Marinomonas and its application

[0034] The genus Marinomonas has abundant proteins related to frost resistance, osmotic pressure resistance, carbohydrate enzymes and various secondary metabolites. In the present invention, a Gram-negative, rod-shaped, non-flagellated, strictly aerobic bacterium isolated from red algae (seaweed) in the coastal area of ​​Weihai, China, is numbered as E8. According to polyphasic taxonomic studies, strain E8 belongs to a new species of the genus Marinomonas, named Marinomonas algarum.

[0035] The strain provided by the invention is a new strain of Marinomonas sp., named as Marinomonas sp. E8, and has the function of high-yield ectoine.

[0036] Specifically, the physiological characteristics of the Marinomonas sp. E8 of the present invention are as follows: the cells are Gram-negative, rod-shaped, flagella-free, catalase and oxidase-negative, strictly aerobic bacteria, about 0.2-0.4 μm wide and 1.0-2.5 μm long. After culturing on 2216 medium at 28°C for 48 hours, the colonies are white, round and smooth. In the presence of 0.5-8% NaCl (w / v; optimum 1-3%), temperature 15-35°C (optimum 25-30°C), pH 6.0-9.0 (optimum 6.5-8.0). Strain E8 has the ability to oxidize D-mannose and D-mannitol, and has the ability to utilize citrate, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, ferric citrate of esculin, D-lyxose, D-tagatose, 5-ketogluconate potassium, and weaker ability to assimilate arabinose and D-somnitr. The DNA G+C content is 42.8 mol%. The main quinone is Q-8, and the main fatty acid is C 16:0 ,summedfeature 3(C 16:1 ω7c and / or C 16:1 ω6c) and summed feature 8(C 18:1 ω7c and / or C 18:1 ω6c). The main polar lipids are phosphatidylglycerol and phosphatidylethanolamine. This strain is E8 (=KCTC 92201 T =MCCC 1K07070 T ), isolated from the red alga Gelidium amansii.

[0037] The 16SrRNA sequence (GenBank NO.OK444097, SEQ ID NO.1) is as follows:

[0038]

[0039]

[0040] Microbial Agents

[0041] The active substance of the present invention (Marinomonas algarum) can be prepared into a bacterial preparation by conventional methods, for example, solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with the active substance, microcapsules in polymers.

[0042] These preparations can be produced by known methods, for example, microbial fermentation.

[0043] In the present invention, the "bacterial agent" contains the active ingredient of Marinomonas algarum.

[0044] The bacterial agent of the present invention can be made into a mixture with other substances, such as cysteine, glutathione, butylated hydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), and vitamin E to be present in their commercial preparations or in the dosage forms prepared from these preparations.

[0045] In addition, the bacterial agent of the present invention can also be prepared into a mixture with a synergist and exist in their commercial preparations or in dosage forms prepared from these preparations. These synergists are compounds that enhance the action of active bacteria. Since the active bacteria themselves are active, it is not necessary to add a synergist.

[0046] The bacterial agent of the present invention may be in various dosage forms. From the standpoint of ease of preparation and application, preferred enzyme preparations include sprays and solution preparations.

[0047] The bacterial agent of the present invention generally contains 1×10-1×10 15 cfu / mL or cfu / g Marinomonas spp., preferably 1×10 4 -1×10 9 cfu / mL or cfu / g Marinomonas algarum, based on the total volume or total weight of the bacterial preparation. The concentration of the strain of the present invention in the commercial preparation or dosage form can vary within a wide range.

[0048] Strain preservation

[0049] The strain Marinomonas sp.E8 of the present invention (the same as the deposited name) was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on June 6, 2022, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.25012.

[0050] Morphological, physiological, biochemical and chemotaxonomic characteristics showed that Marinomonas sp.E8 was a new species of the genus Marinomonas. Comparative genome analysis also showed that Marinomonas sp.E8 was a new species of the genus Marinomonas, so the new species was named Marinomonas algarum.

[0051] The main advantages of the present invention include:

[0052] (1) The present invention is the first to discover a new bacterial species, Marinomonas algarum, from red algae, which has the ability to produce ectoine.

[0053] (2) The present invention is the first to isolate a new strain from red algae, namely, Marinomonas sp. E8 strain.

[0054] (3) The present invention conducted a comparative genome analysis of secondary metabolites and homologous proteins on 30 reference genome sequences of the genus Marinus, and measured the fermentation level of the secondary metabolite ectoine.

[0055] In this study, a Gram-negative, rod-shaped, non-flagellated, strictly aerobic bacterium was isolated from red algae (seaweed) off the coast of Weihai, China, and the strain number was E8. The optimal growth temperature of strain E8 is 25-30°C, the optimal growth pH is 6.5-8.0 and the optimal growth salinity is 1-3% (w / v) sodium chloride. The DNA G+C content of this strain is 42.8 mol%. The main quinone type is Q-8, and the main fatty acid is C 16:0 , summed feature 3 and summed feature 8. The main polar lipid types are phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). According to polyphasic taxonomic studies, strain E8 belongs to a new species of the genus Marinomonas, named Marinomonas algarum. The type strain is E8 (= KCTC92201 T =MCCC 1K07070 T ).

[0056] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where no specific conditions are indicated are usually performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in Microorganisms: A Laboratory Manual (James Cappuccino and Natalie Sherman, Pearson Education Press), or according to the conditions recommended by the manufacturer.

[0057] Unless otherwise specified, the materials and reagents used in the examples are commercially available products.

[0058] Materials and methods

[0059] Isolation, cultivation and preservation

[0060] The experimental samples were collected from the coast of Xiaoshi Island, Weihai, China (37.5°N, 122.1°E) and brought back to the laboratory in sterile sampling bags. The samples were broken into small pieces and homogenized, and then 1g of the homogenate was weighed out and added to 9ml of sterilized seawater and mixed evenly for 10 -1 . After gradient dilution to 10 -2 and 10 -3 , 100 μl of each of the three dilutions was spread on marine agar 2216 (MA, Becton Dickinson) and cultured at 25°C for 5 days. Strain E8 was streaked onto new MA medium (Xu et al. 2020), and the strain was stored at -80°C in 20% (v / v) glycerol supplemented with 1% (v / v) sodium chloride. The reference bacteria was Marinomonas communis JCM20766 T and Marinomonas pontica DSM17793 T .

[0061] 16S rRNA gene sequence and phylogenetic analysis

[0062] To determine the taxonomic status of strain E8, the 16S rRNA gene sequence was amplified by PCR using two universal primers, 27f and 1492r (Liu et al. 2014). The purified PCR product was ligated with the pGM-T vector and transformed into Escherichia coli DH5α cells. The 16S sequence of strain E8 was sent to BGI Co., Ltd. (Wuhan, China) for detection and sequencing using the ABI 3730XL system. The amplified 16S rRNA gene sequence of strain E8 was submitted to the GenBank database, and the BLAST algorithm of NCBI (https: / / www.ncbi.nlm.nih.gov) and EzTaxon server (http: / / www.ezbiocloud.net / ) (Yoon et al. 2017) was used to calculate the 16S rRNA gene sequence similarity. The phylogenetic tree was constructed using the neighbor-joining, maximum likelihood, and maximum parsimony algorithms using MEGA version 7.0 (Kumar et al. 2016). Genetic distances were calculated using the Kimura two-parameter model, and tree topology was assessed by bootstrap analysis with 1000 replicates ( Felsenstein 1985 ).

[0063] Genome sequencing and functional analysis

[0064] The genomic DNA of strain E8 was extracted from MB (Becton Dickinson) medium after 48 h of culture using a bacterial DNA kit (Omega) according to the manufacturer's recommended method, and the purified DNA was sent to Beijing Novo Biotechnology Co., Ltd. To ensure the accuracy of 16S rRNA, the complete gene sequence of 16S rRNA was obtained from the draft genome using the RNAmmer 1.2 server (http: / / www.cbs.dtu.dk / services / RNAmmer / ) and compared with the PCR amplification results. The chromosomal DNA G+C content was calculated using the genome sequence. The genome components were predicted using the GeneMarkS program (Besemer et al. 2001), tRNAscan-SE software (version 1.3.1) (Lowe and Eddy 1997), rRNAmmer software (version 1.2) (Lagesen et al. 2007), Rfam database (Gardner et al. 2009), IslandPath-DIOMB program (Hsiao et al. 2003), PHAST software (version 2.3) (Zhou et al. 2011) and CRISPRFinder (Grissa et al. 2007) to predict the relevant coding genes tRNA, rRNA, snRNA, Genomics Islands, phage protozoa and clustered regularly interspaced short palindromic repeats (CRISPR). After Blast, the whole genome was searched (E-value less than 1e -5, the minimum alignment length percentage was greater than 40%) for predicting gene function by using Gene Ontology (GO) (Ashburner et al. 2000), Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al. 2015), Clusters of Orthologous Groups (COG) (Galperin et al. 2015), using Rapid Annotations using Subsystem Technology (Rastekenari et al.) server (Aziz et al. 2008) and Transporter Classification Database (TCDB) (Saier et al. 2014). To further examine the taxonomic relationship between strain E8 and members of the genus Marinomonas, phylogenomic analysis was performed based on the bac120 marker gene set by GTDB-Tk (Chaumeil et al. 2020), using FastTree (Price et al. 2010) to reconstruct the gene sequence (JTT+CAT parameters) and IQ Tree (Trifinopoulos et al. 2016) analysis using the LG+F+I+G4 model and 1000 bootstrap replicates.

[0065] Morphological, physiological and biochemical analyses

[0066] Strain E8 was transferred to MA medium and cultured at 28°C for 48 h to observe its morphology and physiological characteristics. Cell morphology, size, and the presence of flagella were observed using light microscopy (E600, Nikon) and transmission electron microscopy (JEM-1200; JEOL) in the State Key Laboratory of Biofibers and Eco-Textiles (Qingdao University, China). Gram staining was performed according to the method of Smibert and Krieg (1994). 0.3% agar was added to MB liquid medium, and its motility was detected by the hanging drop method according to the method of Bernardet et al. (2002). Strain E8 cells were placed in MA medium containing 0.1% (w / v) KNO3 in an anaerobic jar for 14 days, while the control group did not add KNO3. Its growth under anaerobic conditions (10% H2, 10% CO2, and 80% N2) was measured. The plates of strain E8 with three zones were placed in incubators at 4, 10, 15, 20, 25, 28, 30, 33, 37, 40 and 45°C, with 5 parallels for each gradient, and the growth of each plate was observed. Salinity detection: The original 2216E culture medium formula was used to prepare artificial seawater without NaCl (per liter: 3.2g MgSO4, 2.2g MgCl2, 1.2g CaCl2, 0.7g KCl, 0.2g NaHCO3), and different concentrations of NaCl were added. The gradient was set to 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% (w / v), and the OD was measured every 12h using a spectrophotometer. 600 The absorbance of bacteria was measured and the growth of colonies was observed. pH test was performed by adding different buffers to 2216 (MB; BD), adjusting the pH to 5.5 and 6.0 with MES, 6.5 and 7.5 with PIPES, 7.5 and 8.0 with HEPES, 8.5 with Tricine and 9.0, 9.5 and 10.0 with CAPSO, and using a spectrophotometer OD 600 The absorbance of the bacteria was measured. Agar, starch, cellulose, alginate, casein and lipids (Tween 20, 40, 60 and 80) and nitrate reduction were tested as described by Tindall et al. (2007). API 20E, API ZYM, API 50CHB and oxidase kit (bioMérieux) kits (all from BioMérieux, France) and Biolog GenIII microPlates (except for those adjusted to 3% salinity) were used for each physiological or biochemical test. All tests were performed at least twice with reference bacteria. The susceptibility to antibiotics was determined according to the 0.5 McFarland standard method at 28°C. Cultivation and reading of the inhibition zone diameter strictly followed the CLSI standards (CLSI 2012).

[0067] Chemotaxonomic characterization analysis

[0068] To analyze the cellular fatty acids, respiratory quinone composition and polar lipid characteristics of strain E8 cells, E8 and two other reference strains were grown in MB medium at 28 °C for 3 days and freeze-dried. Saponification, methylation and extraction of cellular fatty acids were performed according to the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B) equipped with an Agilent HP6890 gas chromatograph (Sasser 1990). The nomenclature and percentage of fatty acids were determined using MIS standard software and the TSBA40 database. Respiratory quinones were separated by thin layer chromatography after extraction from 300 mg of lyophilized cell material with a chloroform / methanol (2:1, v / v) mixture (Tindall et al. 2007). Subsequently, the content of each quinone type was analyzed by HPLC according to the method of Kroppenstedt (1982). Polar lipids were extracted with chloroform / methanol / water solution (65:25:4) and a second extraction with chloroform / methanol / acetic acid / water (80:12:15:4) and separated by two-dimensional silica gel thin layer chromatography. The lipid material was stained with 10% molybdenum phosphate solution (total lipids), molybdenum blue solution (phosphate), α-naphthol sulfate solution (carbohydrate) and ninhydrin (amine). The specific experimental process was carried out according to the methods described by Minnikin et al. (1984) and Tindall et al. (2007).

[0069] Comparative genomic and pan-genomic analyses

[0070] Comparative genomics studies were performed using 29 reference genomes of other Marinomonas species and M. algarum E8 from the GenBank database. The genome of this strain was analyzed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). To determine the genomic distance between each genome, MASH distances were calculated using MASH (Ondov et al. 2016). Whole-genome pairwise comparisons of average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated according to the Konstantinidis and Tiedje script (http: / / enve-omics.gatech.edu / ). Digital DNA-DNA hybridization (dDDH) values ​​were determined using the Genome-to-Genome Distance Calculator (GGDC2.1) (http: / / ggdc.dsmz.de).

[0071] Protein sequences were clustered and compared using Cd-hit (Li and Godzik 2006) and aligned using the MAFFT method (Katoh et al. 2002). Protein sequences were conserved to the corresponding codon alignment using trimAl and automatically aligned for trimming (Capella-Gutiérrez et al. 2009). Then, FastTree constructed a single-copy protein tree (Price et al. 2010). To identify conserved single-copy gene families, the genomes of strain E8 and 29 reference strains were analyzed using OrthoFinder (Emms and Kelly 2019), and a phylogenetic tree of single-copy orthologue proteins was constructed using FastTree following default parameters (Price et al. 2010).

[0072] Pan-genome analysis was performed using the Bacterial Pan Genome analysis tool BPGA (Chaudhari et al. 2016) to identify core genes, accessory genes, and unique genes. The pipeline generated an outgroup-free phylogenetic tree based on the pan-matrix data for the pan-genome tree to better demonstrate the relationships among Marinomonas species. In addition, the protein sequences of strain E8 and 11 orthologous clusters isolated from marine plants, including M. agarivorans QM202, were analyzed using the OrthoVenn2 web server (Xu et al. 2019). T ,M.alcarazii CECT 7730 T ,M.algicola SM1966 T ,M.aquiplantarum CECT 7732 T ,M.balearica CECT 7378 T ,M.colpomeniae SM2066 T ,M.foliarum CECT 7731 T ,M.pollencensis CECT 7375 T ,M.posidonica IVIA-Po-181 T ,M. rhizomae IVIA-Po-145 T andM. spartinae CECT 8886 T .

[0073] To further compare genome functions and metabolism, all 30 reference genome sequences were annotated using Prokka (Seemann 2014). The COG database (Galperin et al. 2015) was used to determine homologous protein groups and predicted functions, and the results for each strain were clustered into a relative abundance heat map. -5 COG annotation was selected when the reference strains were selected. The genomes of the reference strains were analyzed using antiSMASH version 6.0 (Blin et al. 2021) with stringent detection criteria to predict biosynthetic gene clusters (BGCs) (default parameters). Stacked plots depicting the number of each BGC type were generated using the Rstudio package ggplot2 (Villanueva and Chen 2019). The BAGEL4 web server (van Heel et al. 2018) was used to further explore potential RiPPs and bacteriocins in all reference strains of the genus Marinomonas.

[0074] Example 1 Morphological, physiological and biochemical characteristics

[0075] The cells of strain E8 are Gram-negative, rod-shaped, about 0.2-0.4 μm wide and 1.0-2.5 μm long, without flagella ( Figure 1 The optimal growth temperature is 25-35°C, the pH value is 7.0-8.0, and the salinity range is 1.0-4.0%. The main differences in the biochemical characteristics of strain E8 and the reference bacteria are summarized in Table 1.

[0076] According to the results of API ZYM and API 20E, strain E8 has alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamine, valine arylamine, acid phosphatase, naphthol-AS-BI-phosphohydrolase activities. Strain E8 did not hydrolyze cellulose, starch, alginate, Tween 40, 60, 80, and was negative for nitrate reduction. The results of antibiotic sensitivity test are as follows: E8 is sensitive to penicillin (10), streptomycin (10), tobramycin (10), gentamicin (10), ampicillin (10), ofloxacin (5), carbenicillin (100), ceftriaxone (30), norfloxacin (10), cefotaxime (30), clarithromycin (CLR) (15), polymyxin B (300), chloramphenicol (30); intermediate: neomycin (30), rifampicin (5), kanamycin (30), erythromycin (15); resistant: vancomycin (VA) (30), tetracycline (30), lincomycin (2).

[0077] Table 1. Differential characteristics of strain E8 and its closely related species

[0078]

[0079]

[0080] Strains: 1, E8; 2, M. pontica DSM 17793 T ; 3, M. communis JCM 20766 T Unless otherwise stated, all data are from this study. + positive, - negative, w weakly positive.

[0081] Example 2 Phylogenetic and Phylogenomic Analysis

[0082] The 16SrRNA gene sequence (1425 bp) of strain E8 was amplified by PCR and contained a complete 16SrRNA genome sequence. The blast results of NCBI showed that strain E8 was similar to the reference strain M. vulgaris A79. T (98.4%) and M. pontica 46-16 T The similarity between strain E8 and Marinomonas communis JCM 20766 was the highest (97.5%). T The proportion of strain E8 and A79 was 94.9%. Based on the maximum likelihood phylogenetic tree of 16srRNA gene, strain E8 and A79 T A separate branch was formed on the close relationship ( Figure 2 ). In addition, the IQ phylogenetic tree based on the genome sequence showed that strains E8 formed a branch with A79 T The strain E8 was distinguished from other members of the genus and confirmed to be a member of the genus Marinomonas ( Figure 3 ).

[0083] Figure 2 Maximum likelihood phylogenetic tree of strain E8 16S rRNA gene sequences of other members of the Oceanospirillaceae. All strains of the genus Marinomonas have been identified. Branch nodes (ML / NJ / MP) show bootstrap values ​​(greater than 0%) for 1000 replicates. T (GenBank accession number MG706141) is the outgroup. Bar, 0.01 substitution at each nucleotide position.

[0084] Example 3 General genomic features

[0085] The draft genome sequence of strain E8 was assembled into 79 contigs with a total length of 3,285,337 bp, an N50 value of 126,031 bp, an L50 value of 8, and an average coverage of 150×. The longest and shortest sequences were 379 bp, 893 bp, and 618 bp, respectively. The DNA G+C content was 45.1 mol%, which was in the middle position of most closely related species. A total of 3085 genes were predicted, including 2988 protein-coding genes, 97 coding RNAs, including 8 5SRNAs, 4 16SRNAs, 4 23SRNAs, 77 RNAs, and 4 ncRNAs. The genome sequence of strain E8 included 31 long interspersed nuclear elements (LINEs), 23 short interspersed nuclear elements (SINEs), 4 genomic islands (GIs), and 2 CRISPR-related genes.

[0086] According to the predicted genome functions, strain E8 has annotated complete glycolysis, gluconeogenesis, citric acid cycle (TCA cycle) and pentose phosphate pathways, and is capable of central carbohydrate metabolism and UDP-N-acetyl-D-glucosamine biosynthesis, which is consistent with M. vulgaris A79. T and M. communis JCM 20766 T Same. In strains E8 and A79 T Succinate dehydrogenase, cytochrome bd ubiquitin oxidase, cytochrome o ubiquitin oxidase, and F-type ATPase were predicted in JCM 20766. T The cytochrome c oxidase and cytochrome bc1 complex respiratory units are absent from the genomes of strains E8 and A79 T The genome lacks the ackA gene and cannot be like JCM 20766 T The strain A79 converts acetyl-CoA into acetate as if there were a complete carbon fixation pathway. TThe strain E8 contains cysNC, cysN, cysD, cysNC, cysC, cysH, cysJ, and cysI genes, which can convert sulfate into sulfide, but the strain E8 genome does not have the cysC gene and can only convert sulfite into sulfide. Genome analysis predicted that strain E8 metabolizes multiple amino acids, including serine, threonine, valine, isoleucine, leucine, lysine, ornithine, arginine, proline, and tryptophan. Regarding cofactor and vitamin metabolism, E8 has complete synthetic pathways for pyridoxal-P (EC1.4.3.5), NAD (EC 6.3.5.1), pantothenate (EC 6.3.2.1), coenzyme A (EC 6.3.2.5), biotin (EC2.8.1.6), lipoic acid (EC 2.8.1.8), molybdenum cofactor (EC 2.10.1.1), heme (EC 4.99.1.4), heme (EC 1.3.5.3), and cobalamin (EC 6.3.1.10). The genomes of these three strains contain multiple genes responsible for choline uptake and betaine biosynthesis, including choline dehydrogenase (betA), betaine-acetaldehyde dehydrogenase (betB), and glycine betaine ABC transport system permease (ProU) (betaine is an osmoprotectant). In addition, all genes mediating the synthesis of phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were found in the genome of strain E8, which were similar to other strains of the genus Marinomonas.

[0087] Example 4 Chemotaxonomic Characteristics

[0088] The test results showed that the main fatty acid (greater than 10% fatty acid component) of strain E8 was C 16:0 ,summedfeature 3(C 16:1 ω7c and / or C 16:1 ω6c) and summed feature 8(C 18:1 ω7c and / or C 18:1 ω6c). The ratio of summed feature 8 was 47.8%, which was the most abundant cellular fatty acid in E8 and also in strain M. pontica DSM17793. T and M. communis JCM 20766 T The most abundant fatty acids in strain E8 were 35.3% and 45.7%, respectively. 17:0 The proportion of the other two strains was 3.1%, and the proportion of the other two strains was less than 0.5%. The summed feature 8 in the fatty acid composition of strain E8 was significantly higher than that of M. pontica DSM 17793 T, but with M. communis JCM 20766 T Almost. The fatty acid profile of strain E8 and the reference strain M. pontica DSM 17793 T and M. communis JCM 20766 T The results are shown in Table 2. The polar lipids were mainly composed of phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and three unknown lipids (L1-L3), which was similar to other model strains (PG and PE were the main polar lipids ( Figure 4 )). The isoprenoid quinone (respiratory quinone) detected in strain E8 was consistent with that in other model strains and was Q-8.

[0089] Table 2. Fatty acid composition of strain E8 and control strain (%)

[0090]

[0091] Strains: 1, E8; 2, M. pontica DSM 17793 T ; 3, M. communis JCM 20766 T . Values ​​are percentages of total fatty acids. Major fatty acids (>10%) are in bold; fatty acids with less than 0.5% in all strains are omitted. TR, trace content <0.5%) —, not detected. The branched-chain fatty acid Anteiso-C was found in 3 strains. 15:0 、Iso-C 16:0 and Anteiso-C 16:0 , molecular weight is less than 0.5%.

[0092] *Summed Features refers to two or three groups of fatty acids that cannot be separated by GLC in the MIDI system. Summed Feature 3, C 16:1 ω7c and / or C 16:1 ω6c;Summed Feature 7,C 19:1 ω7c and / orC 19:1 ω6c;Summed Features 8,C 18:1 ω7c and / or C 18:1 ω6c.

[0093] Example 5 Comparative genome analysis

[0094] The genomic distance results between the genome sequences showed that strain E8 and M. vulgaris A79 T The closest to M.agarivorans QM202 T、M.flavescens ANRC-JHZ47 T and M. posidonica IVIA-Po-181 T Farther distance ( Figure 5 ). strain E8 and common M. gargaris A79 T The ANI and AAI between strain E8 and M. vulgaris A79 were 78.7% and 84.3%, respectively, which are lower than the species boundaries of 95-96% recommended by Richter and Rossello-Mora and 85-90% proposed by Qin et al. T The dDDH value of is also low (22.0%), which is lower than 70% of species (Goris et al.; Meier-Kolthoff et al.). Figure 6 Pairwise genome comparisons at the nucleotide and protein levels (ANI and AAI values) are shown, which indicate that strain E8 is a new species of the genus Marinomonas.

[0095] Single copy core protein tree showing strain E8 and M. vulgaris A79 T Close branches formed with the trunk formed by all 30 reference strains ( Figure 7 ). M.agarivorans QM202 T and M.algicola SM1966 T The longer branch lengths of the phylogenetic tree may indicate a more distant phylogenetic relationship. The phylogenetic tree constructed from single-copy homologous proteins also confirmed the tree ( Figure 7 ). The results of single-copy gene families showed that a total of 6806 subgroups were clustered, of which 104049 genes were in the positive subgroups and 3629 genes were unassigned, and a total of 1379 subgroups were clustered in all 30 strains.

[0096] The results of pan-genome homologous groups (POGs) detection of 30 strains of Marinomas showed that strain E8 had 959 core genes, 1661 accessory genes, 219 unique genes and 10 completely missing genes. The core pan-genome map showed that the pan-genome of Marinomas can be considered "open" ( Figure 8 ). The analysis pipeline generates a pan-genome phylogenetic tree without outgroups based on the pan-matrix data, which highlights distinct groups and is confirmed in the core genome tree ( Fig. 9,10). According to KEGG annotation, strains of the genus Marinus are active in amino acid metabolism, carbohydrate metabolism, membrane transport, metabolism of cofactors and vitamins, and biodegradation and metabolism of xenobiotics. A large number of unique genes are related to amino acid metabolism, carbohydrate metabolism, and xenobiotic degradation ( Fig.11 ). The selected model strains (12 species) related to marine plants formed a total of 5297 clusters, of which 3916 were homologous clusters (containing at least two species) and 1381 were single-copy gene clusters (Table 3). Five of the genomes had a total of 1510 protein coding regions, and the genome of strain E8 encoded 2904 proteins, of which 2,604 were clustered and 261 were single-copy ( Fig.12 ).

[0097] Table 3. Summary of proteins, clusters, and monocytes in the genomes of 12 strains

[0098]

[0099]

[0100] Strain E8, M. aquimarina CECT 5080 T and M. vulgaris A79 T The strains had a lower frequency of carbohydrate transport and metabolism (COG functional category G), which may be related to the nutrient-poor environment in which they live. T In comparison, strain E8 had a higher number of proteins related to genetic information transmission and transcription in categories J, X, and D ( Fig.13 ). Proteins related to the biosynthesis of secondary metabolites were observed in all strains. Based on the antiSMASH annotation, abundant BGCs were predicted in the genome sequences of 30 strains. Overall, gene clusters encoding exotoxins, nonribosomal polyketide synthetases (NRPS), β-lactones, and some other undefined ribosomal synthesized and post-translationally modified peptide products (RiPP) (Pipp analogs) were predicted in the genomes of most strains. The Pipp analogs in strain E8 were annotated as bacteriocins, which were also annotated in Marinomonas primoryensis MPKMM3633, Marinomonas pollencensis CECT 7375 and Marinomonas pollencensis CECT 8887 with high gene similarity. MiBiG alignment results showed that the bacteriocins of strain E8 had the highest similarity with the compounds lanthipeptides (MiBiG accession number BGC0000554) in Streptomyces NRRL 15998. In addition, M. spartinae CECT 8886T A type I PKS (Polyketide synthase) gene cluster was annotated, which may mediate the biosynthesis of cylindrosporin. According to the prediction of BAGEL4 database, in M. polaris DSM 16579 T ,M.mediterranea MMB-1 T and M.rhizomae IVIA-Po-145 T Lasso peptides were predicted in M. piezotolerans YLB-05; microcins were predicted in M. piezotolerans YLB-05 T and M.algicola SM1966 T are predicted in M. rhizomae IVIA-Po-145; butyricin, garvicin_ML, and bottromycin T ,M.flavescens ANRC-JHZ47 T andM.atlantica Cmf 18.22 T were predicted respectively.

[0101] Example 6 Description of Marinomonas algarum

[0102] The cells are Gram-negative, rod-shaped, flagella-free, catalase and oxidase-negative, strictly aerobic bacteria, about 0.2-0.4 μm wide and 1.0-2.5 μm long. After culturing on 2216 medium at 28°C for 48 hours, the colonies are white, round and smooth. In 0.5-8% NaCl (w / v; optimum about 1-3%), temperature 15-35°C (optimum about 25-30°C), pH 6.0-9.0 (optimum 6.5-8.0). Strain E8 has the ability to oxidize D-mannose and D-mannitol, and has the ability to utilize citrate, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, ferric citrate, D-lyxose, D-tagatose, 5-ketogluconate potassium, and weaker ability to assimilate arabinose and D-suzanol. The DNA G+C content is 42.8 mol%. The main quinone is Q-8, and the main fatty acid is C 16:0 ,summed feature 3(C 16:1 ω7c and / or C 16:1 ω6c) and summed feature 8(C 18:1 ω7c and / or C 18:1 ω6c). The main polar lipids are phosphatidylglycerol and phosphatidylethanolamine. This strain is E8 (=KCTC 92201 T =MCCC 1K07070 T), isolated from the red alga Gelidium amansii.

[0103] Example 7 Study on the production of ectoine by strain E8

[0104] This example studies the ability of strain E8 to produce ectoine. Eectoine is a cyclized amino acid derivative with a molecular formula of C6H10O2N2 and a molecular weight of 142.16. Eectoine has salt resistance, antifreeze, and anti-drying properties, and can protect and stabilize macromolecules such as enzymes, DNA, and lipid membranes in organisms. It has a wide range of applications in the fields of cosmetics, food, and biopharmaceuticals.

[0105] After preliminary optimization, the fermentation conditions for the production of ectoine by strain E8 were:

[0106] The culture was carried out using 2216 liquid culture medium with an initial pH of 7.5-8.0. The NaCl concentration in the culture medium was set to 4% (w / v). The culture was shaken at 28° C. and 120 rpm for 48 hours to obtain a fermentation broth.

[0107] Ectoin standard was purchased from Sigma. HPLC conditions for detecting ectoin:

[0108] Chromatographic column: Eclipse XDB-C18 column (150×4.6mm),

[0109] Mobile phase: acetonitrile: water mixture (5:95, volume ratio),

[0110] Flow rate: 0.8ml / min,

[0111] Detection wavelength: 210nm,

[0112] Column temperature: 30℃.

[0113] The content of ectoine in the samples was calculated based on the standard curve of the standards.

[0114] Determination of ectoine fermentation level:

[0115] The fermentation broth was centrifuged to collect the bacterial cells, and the bacterial cells were quickly washed twice with sterile water, and the bacterial cells were filtered through a microporous filter membrane each time.

[0116] Add 10 times the weight of anhydrous ethanol to the wet cells and extract at 50°C for 3 hours. After the extraction is completed, filter the filtrate, centrifuge the filtrate at high speed and use it as the sample for high-performance liquid chromatography detection. According to the HPLC test results, calculate the yield of tetrahydropyrimidine in the fermentation broth.

[0117] HPLC analysis showed that the average concentration of ectoine in the six fermentation broths of strain E8 was 12.4 g / L.

[0118] Example 8 Preparation of Ectohydropyrimidine by fermentation of wastewater from gelatin production

[0119] The wastewater from the gelatin industry contains a large amount of pollutants such as oil, protein, etc., and is a wastewater with high organic concentration. The pH of the wastewater from the gelatin industry is high, the organic content is high, and the treatment cost is extremely high.

[0120] The gelatin industrial wastewater used in this example was collected from the water treatment workshop of Weihai Yinhe Biotechnology Co., Ltd. After measurement, its characteristic indicators are as follows:

[0121] The pH is 9.6, COD is 34085 mg / L, BOD is 20561 mg / L, total phosphorus is 4832 mg / L, ammonia nitrogen is 5637 mg / L, and the sodium chloride content is 4.2%.

[0122] The gelatin industrial wastewater was adjusted to pH 8.0 and sterilized at 60°C for 2h for later use.

[0123] Strain E8 seed liquid fermentation:

[0124] The culture was carried out using 2216 liquid culture medium with an initial pH of 7.5-8.0 and a NaCl concentration of 4% (w / v). The culture was shaken at 28° C. and 120 rpm for 36 hours to obtain a seed solution.

[0125] The seed liquid was added into the above-mentioned gelatin industrial wastewater at a volume ratio of 10%, and cultured at 28°C and 120 rpm with shaking for 48 hours.

[0126] After the fermentation was completed, the bacteria were obtained by centrifugation, and the COD in the fermentation supernatant was measured to be 4352 mg / L, BOD to be 454 mg / L, total phosphorus to be 186 mg / L, and ammonia nitrogen to be 64 mg / L. The contents of various organic pollutants were significantly reduced.

[0127] HPLC detection showed that the average concentration of ectoine in the 6 groups of gelatin industrial wastewater fermentation broths of strain E8 reached 26.3 g / L, which was significantly higher than the ectoine yield obtained by fermentation in artificial 2216 medium. The reason for this was that the chemical stress factors in gelatin industrial wastewater stimulated the ectoine metabolism level of strain E8 to protect the normal growth of the bacteria.

[0128] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A Marinomonas, characterized in that The Marinomonas is Marinomonas ( Marinomonas sp. ) E8 strain, whose deposit number is CGMCC NO.25012.

2. A microbial agent, characterized in that: The microbial agent comprises: the Marinomonas described in claim 1 and / or its fermentation broth.

3. The microbial agent according to claim 2, characterized in that: In the microbial agent, the content of the sea mononas is 1×10 -1 Up to 1×10 15 cfu / mL or cfu / g, based on the total volume or total weight of the microbial inoculant.

4. The microbial agent according to claim 3, characterized in that: In the microbial agent, the content of the sea mononas is 1×10 4 Up to 1×10 9 cfu / mL or cfu / g, based on the total volume or total weight of the microbial inoculant.

5. The microbial agent according to claim 2, characterized in that: The microbial agent contains 0.0001-99 wt % of the Marinomonas and / or its fermentation liquid, based on the total weight of the microbial agent.

6. The microbial agent according to claim 5, characterized in that: The microbial agent contains 0.1-90wt% of the Marinomonas and / or its fermentation liquid, based on the total weight of the microbial agent.

7. The microbial agent according to claim 2, characterized in that: The dosage form of the microbial agent is selected from the following group: solution, suspension or powder.

8. A use of the Marinomonas according to claim 1 or the microbial agent according to claim 2, characterized in that: Used for fermentation production of tetrahydropyrimidine or for wastewater treatment.

9. The use according to claim 8, characterized in that The wastewater is gelatin industrial wastewater.

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