Compound ABAGG and application thereof

Abstract

The invention discloses a compound ABAGG and application thereof. The present invention relates to a micromonospora strain produced from a micromonospora strain that converts glycerol and glycine. The micromonospora strain is deep micromonospora profusii TRM 95458, the micromonospora profusii TRM 95458 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on August 2, 2022, and the preservation number of the micromonospora profusii TRM is GDMCC NO: 62600. The strain is used for producing compounds containing glycerol groups such as ABAGG, waste is turned into wealth, and the strain has a good industrial application prospect.

Description

[0001] This invention is a divisional application with Chinese patent application number 202211744304.X, the invention name is "A strain of actinomycetes for converting glycerol and glycine", and the application date is December 25, 2022. Technical Field

[0002] The present invention relates to the field of industrial microorganisms, and in particular to an actinomycete which utilizes glycerol as the sole carbon source to ferment and produce metabolites containing glycerol groups, more particularly to Micromonospora profunda and its application in industry, agriculture and medicine. Background Art

[0003] Microorganisms can use glycerol to convert and produce fine chemicals, among which Klebsiella pneumoniae, Citrobacter freundii and Clostridium butyricum have been studied more (Sun YQ, Shen JT, Yan L, et al. Advances inbioconversion of glycerol to 1,3-propanediol: prospects and challenges [J]. Process Biochemistry, 2018, 71: 134-146.). In recent years, the use of various microorganisms to convert crude glycerol to produce some fine chemicals has attracted the attention of scholars at home and abroad. Qi Xianghui et al. provided a microbial co-culture system, a method for producing 1,3-propanediol by fermenting glycerol, which improved the conversion rate of glycerol and the yield of 1,3-propanediol and reduced the production cost (CN111394396B,). Zhao Zongbao et al. used an extracellular cell-free system or in microorganisms to catalyze the production of 1,3-propanediol from biodiesel byproduct glycerol, significantly reducing the production cost of 1,3-propanediol and improving the product yield (CN110964753B).

[0004] Glycerol is an inevitable byproduct in the production process of biodiesel transesterification, and its output is huge with the development of the biodiesel industry. Microbial conversion has the characteristics of mild conditions, simplicity and easy operation, which makes the application of glycerol in the fermentation field widely concerned (Chilakamarry CR, Sakinah AM, Zularisam AW, et al. Bioconversion of glycerol into biofuels—opportunities and challenges [J]. BioEnergy Research, 2022, 15 (1): 46-61.). Using microbial cell factories to convert renewable biomass resources to produce fine chemicals has important industrial, agricultural and medical application value (Jathanna HM, Rao CV. Using Aspergillus ochraceus, a Native Fungus, to Convert Biodiesel-Derived Crude Glycerol to Single Cell Oil for Commercial Applications [J]. Waste and Biomass Valorization, 2022, 13 (6): 2831-2845.). Summary of the invention

[0005] The present invention isolated a strain of Micromonospora profundi TRM95458 from the chickpea rhizosphere soil in Mulei County, and explored its secondary metabolites, and found that the strain can use glycerol as the only carbon source to produce a new structural compound ABAGG (i.e., compound B), as well as compounds A, D, and E. The structural formula is as follows:

[0006]

[0007] Therefore, the technical problem to be solved by the present invention is: to provide a micromonospora that produces ABAGG, which is Micromonospora profundi TRM95458, which was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on August 2, 2022 (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology), with a preservation number of GDMCC NO: 62600, classified and named: Micromonospora profundi, and survived after testing.

[0008] The application of Micromonospora profunda in the conversion of glycerol and glycine is characterized in that it uses glycerol as the sole carbon source to ferment and produce metabolites containing glycerol groups, which are used to convert renewable biomass resources to produce fine chemicals and are used in industry, agriculture and medicine.

[0009] The present invention provides the application of the Micromonospora profunda in the production of ABAGG. The application is characterized by comprising the following steps: culturing the Micromonospora under suitable conditions to obtain a fermentation broth; separating and preparing ABAGG from the fermentation broth, wherein the fermentation medium for culturing the actinomycetes uses glycerol as the sole carbon source and glycine or the like as a nitrogen source.

[0010] The culture medium formula and culture conditions of the deep micromonospora fermentation are as follows: seed liquid culture medium: starch 10g, TSB 3g, glucose 4g, yeast extract 4g, malt extract powder 10g, trace elements 1mL, agar 17g, and pH 7.0 is adjusted. The solid plate bacteria cake (diameter 1cm) is inoculated into 500mL and 150mL of seed culture medium (3-5 cakes are added to each bottle), and cultured at 30°C and 180rpm for 5 days. Fermentation liquid culture medium: glycerol 20mL, peptone 10g, glycine 10g, yeast extract powder 10g, trace element liquid 1mL, and pH 7.0 is adjusted. Inoculate in the fermentation medium at an inoculum of 4%, and culture on a shaker at 30°C and 180rpm for 7 days.

[0011] The method for separating and preparing ABAGG from a fermentation broth is specifically as follows: after the fermentation broth is centrifuged, the broth is wet-loaded onto a D101 macroporous resin chromatographic column, and then impurities such as sugars, salts and water-soluble proteins are removed with 4 times the column volume of distilled water, and then eluted with 30% to 80% methanol; the 30% to 80% methanol eluate is combined and concentrated under reduced pressure on a rotary evaporator to obtain a fermentation broth crude extract.

[0012] The method for separating and purifying ABAGG from the fermentation broth further comprises the following steps: drying the crude extract of the fermentation broth and dissolving it with methanol, passing it through an ODS column, eluting it with 40%, 60%, and 80% methanol respectively, collecting 80% of the methanol eluate and concentrating it into an extract, and continuing to perform pHPLC to prepare ABAGG.

[0013] The pHPLC preparation conditions are: A: 30% MeOH-H2O (0.075% HCOOH); B: 100% MeOH; flow rate: 10 mL / min; gradient elution conditions: 0-40 min 0-65% B; 40-45 min 65-100% B; 45-50 min 100% B; DAD detector wavelength: 350 nm, collecting the peak with retention time RT = 21.85 min to obtain ABAGG.

[0014] The present invention also provides a compound ABAGG, which has the function of regulating cell osmotic pressure and can improve biological salt tolerance, and its structural formula is as follows:

[0015]

[0016] Furthermore, the present invention also provides the use of the compound ABAGG in regulating cell osmotic pressure or improving biological salt tolerance.

[0017] The present invention has the following beneficial effects: Micromonospora profunda TRM95458 can use glycerol as the sole carbon source to produce metabolites containing glycerol groups, which are used to transform renewable biomass resources to produce fine chemicals and are applied in industry, agriculture and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , Scanning electron microscope photograph of Micromonospora profunda TRM95458 (showing mycelium and round spores).

[0019] Figure 2 , the taxonomic status and phylogenetic tree of Micromonospora profunda TRM95458.

[0020] Figure 3 , HPLC chromatogram of the fermentation product preparation of strain 95458.

[0021] Figure 4 , two-dimensional NMR HMBC long-range correlation diagram of ABAGG.

[0022] Figure 5 , H NMR spectrum of ABAGG.

[0023] Figure 6 , carbon NMR spectrum of ABAGG.

[0024] Figure 7 , NMR HMBC spectrum of ABAGG.

[0025] Figure 8 , HPLC quantitative analysis standard curve of ABAGG.

[0026] Fig. 9 , the yield of ABAGG produced by transformation of Micromonospora australis TRM95458 with different glycerol addition amounts.

[0027] Fig.10 , the yield of ABAGG transformed by Micromonospora australis TRM95458 with different glycine addition amounts.

[0028] Fig.11, the relationship between salt stress and the accumulation of osmotic substance ABAGG in Micromonospora australis TRM95458. DETAILED DESCRIPTION

[0029] The present invention is further illustrated below through detailed descriptions of specific implementation modes, but this does not limit the present invention and is merely an example.

[0030] Example 1: Isolation and classification of Micromonospora profunda TRM95458

[0031] 1 Isolation of Micromonospora profunda TRM95458

[0032] The chickpea rhizosphere soil samples from Mulei County, Xinjiang were collected by dilution plate method with 1 / 10ISP 2 The culture medium (glucose 4g, yeast extract 4g, malt extract powder 10g, trace elements 1mL, agar 17g, pH adjusted to 7.0) was cultured at 28°C to isolate actinomycetes. The strain TRM 95458 was obtained by further screening with glycerol-complex amino acid medium.

[0033] 2 Taxonomic identification of Micromonospora profunda TRM95458

[0034] 2.1 Morphological observation

[0035] Electron microscope observation: solid plate culture, cut off the fungus block (about 0.5cm×0.5cm in size) with a blade, cut off the excess culture medium, and the culture medium with hyphae is thin and of uniform thickness. Use electron microscope to observe and record the hyphae morphology, aerial hyphae and hyphae growth in the base, whether the mycelium produces spores and the arrangement and shape of spores; spore shape and size, etc. Scanning electron microscope photos are shown in Figure 1 .

[0036] Micromonospora profunda TRM95458 grew well on ISP2 medium. The colonies were round and flat, with a dry surface, neat colony edges, abundant aerial hyphae, yellow hyphae in the base, and yellow-brown soluble pigments.

[0037] 2.2 Molecular biological identification of strain TRM95458

[0038] 2.2.1 Extraction of genomic DNA from strain TRM95458

[0039] Collect TRM95458 cells from the culture plate and place them in a 1.5 mL sterile centrifuge tube. Add 480 μL of 1×TE buffer. Add 20 μL of lysozyme (50 mg mL -1 ) and placed in a 37°C water bath overnight. Add 50 μL of 20% SDS to each tube and 5 μL of 20 mg mL-1 Proteinase K was added and placed in a 60℃ water bath for 2h. 550μL of phenol:chloroform:isoamyl alcohol (25:24:1) was added and centrifuged at 12000rpm for 5min. The supernatant was transferred to another centrifuge tube and extracted twice. 300μL of 95% isopropanol and 70μL of sodium acetate (3mol·L -1 ), centrifuge at 12000rpm for 10min, and discard the supernatant. Wash the centrifuged product once with 500μL of 70% ethanol, centrifuge at 12000rpm for 5min, discard the supernatant, and evaporate the ethanol completely. Dissolve the DNA at the bottom with 30μL of sterile ultrapure water, check the quality of DNA extraction by 1% agarose gel electrophoresis, and store the extracted DNA in a -20℃ refrigerator for later use.

[0040] 2.2.2 Amplification of 16S rRNA gene of strain TRM95458

[0041] The 16S rRNA gene fragment in the genomic DNA of actinomycetes was amplified using the universal primers 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3'). The PCR reaction system of 50 μL was: dd H 2 O 34μL, 10× Buffer (buffer containing Mg 2+ )5μL, dNTPs 2.5μL, primer 27F (10μmol·L -1 )2μL, primer 1492R (10μmol·L -1 ) 2 μL, 50% DMSO 2 μL, Taq DNA polymerase 0.5 μL, template DNA 2 μL.

[0042] The PCR reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 2 min, 30 cycles; total extension at 72°C for 8 min. After the reaction was completed, the reaction was detected by 1% agarose gel electrophoresis. The PCR products that met the conditions were sequenced.

[0043] 2.2.3 Comparison and analysis of sequencing results

[0044] The sequencing results were spliced ​​using DNAMAN5.2 software, and the sequences were compared with the effectively published strain sequences in the GenBank database using BLAST. The 16S rDNA gene sequences of the effectively published strains with higher similarity were downloaded, and the phylogenetic tree was constructed for the sequences using MEGA5.0 software to determine the taxonomic status of strain TRM95458.

[0045] 2.2.4 16S rDNA sequence determination results of strain TRM95458

[0046] The sequencing results were spliced ​​using DNAMAN5.2 software, and it was determined that the fragment consisted of 1381 bases. The 16S rDNA sequence of TRM95458 was obtained as follows:

[0047]

[0048]

[0049] 2.2.5 Construction of homology evolution tree

[0050] Through BLAST comparison, sequences with close similarity to the 16S rDNA gene sequences of the actinomycetes to be tested were downloaded from the GenBank database, and multiple sequence alignment of the 16S rDNA gene sequences of actinomycetes was performed using MEGA5.0 software to construct a phylogenetic tree (see Figure 2 The 16S rDNA gene sequences of strain TRM95458 and Micromonospora profundi (100.00% identity) clustered on the same phylogenetic branch, so the micromonospora was identified as Micromonospora profundi TRM95458.

[0051] Example 3: Fermentation and preparation of ABAGG using Micromonospora profunda TRM95458

[0052] Inoculate a small amount of mycelium into ISP 2 The culture medium was cultured at 28°C for 5 days to obtain the seed liquid of Micromonospora profunda TRM95458, and the seed liquid (4% inoculation amount) was inoculated into the fermentation medium for fermentation culture. The fermentation conditions were: rotation speed 180rpm, initial pH 7.0-7.2, liquid volume 150mL, fermentation temperature 30°C, fermentation 7d, and the fermentation liquid was collected. The supernatant was centrifuged and wet-loaded onto a macroporous resin chromatographic column (D101 2.0kg was loaded per 100L of fermentation filtrate), and then eluted with 4 times the column volume of clean water, 30% methanol solution, 80% methanol solution, and 95% methanol solution, respectively; after reduced pressure concentration to remove the solvent, a methanol extract of the fermentation liquid rich in Micromonospora profunda was obtained, and the extract dissolved by methanol ultrasound was passed through an ODS column, eluted with 20%, 40%, 60%, and 80% methanol, respectively, and the eluate was collected for HPLC analysis. Collect 80% of the methanol eluted extract and perform preparative liquid chromatography separation and preparation (see Figure 3), the collected samples were analyzed for the structure of the compound using 1D & 2D NMR techniques to obtain a new compound ABAGG. The HMBC relationship diagram, nuclear magnetic resonance hydrogen spectrum, carbon spectrum, and HMBC spectrum of the new compound ABAGG are shown in Figure 4 , Figure 5 , Figure 6 and Figure 7 The assigned H NMR and C NMR data of ABAGG are shown in Table 1.

[0053] Table 1. ABAGG H NMR and C NMR data

[0054]

[0055]

[0056] Example 4: Quantitative Analysis and Structural Characterization of ABAGG

[0057] ABAGG was quantitatively analyzed by high performance liquid chromatography. The chromatographic conditions were: 4.6 mm x 150 mm C18 column (0.5 μm), 80% methanol-water as mobile phase, flow rate of 1.0 Ml / min, detection wavelengths of 254 nm, 280 nm, 330 nm and 445 nm. The structures of the four compounds obtained are as follows:

[0058]

[0059] Among them, compound B (i.e., ABAGG) is a new structure compound.

[0060] Example 5: Functional assay of glycerol conversion in the production of ABAGG by fermentation of Micromonospora profunda TRM95458

[0061] 1. Prepare the purified ABAGG pure product into 1.0 mg / mL stock solution, and dilute it into a series of concentrations such as 1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156 mg / mL, etc., and determine the standard curve by HPLC through the area normalization method (see Figure 8 ). The standard curve equation of ABAGG is: y = 4.2905x + 0.3875, where x is the relative peak area and y is the content of ABAGG. 2 =0.9929 shows that the linear relationship is good and can be used to determine the content of ABAGG.

[0062] 2. In the fermentation medium (millet 10g, bean sprouts 100g, glucose 10g, peptone 5g, NaCl 2.5g, (NH 4 ) 2 SO 41g, adjust pH 7.0) to add different amounts of glycerol and glycine to obtain different amounts of ABAGG. The results showed that when the amount of glycerol added was 0.1mL and the amount of glycine added was 1g, the ABAGG yield was the highest. Among them, Fig. 9 The yield of ABAGG produced by transformation of Micromonospora profunda TRM95458 with different glycerol addition amounts is shown; Fig.10 Shows the yield of ABAGG produced by transformation of Micromonospora acuminatum TRM95458 with different amounts of glycine added.

[0063] Example 6. Relationship between salt stress and the accumulation of osmotic substance ABAGG by Micromonospora profunda TRM95458

[0064] When 0 g / L, 2.5 g / L, 5 g / L, 10 g / L, 15 g / L, and 20 g / L NaCl were added to the fermentation broth of Micromonospora profunda TRM95458, the yield of ABAGG in the fermentation broth first increased and then decreased with the increase of NaCl concentration. When the NaCl content was 10 g / L, the yield of ABAGG was the highest (e.g. Fig.11 ). This indicates that under higher salt concentrations, Micromonospora profunda TRM95458 improves its salt tolerance by converting glycerol and glycine and accumulating the osmotic pressure regulating substance ABAGG in the cells. ABAGG is a new structure compatible substance with osmotic pressure regulating function.

Claims

1. A compound ABAGG, whose structural formula is as follows: 。 2. The compound ABAGG according to claim 1, characterized in that It is obtained from Micromonospora profunda (GDMCC NO: 62600). Micromonospora profundi ) can be separated.

3. Use of the compound ABAGG according to claim 1 or 2 in regulating cell osmotic pressure or improving biological salt tolerance.

4. Use of the compound ABAGG according to claim 1 or 2 as an osmotic substance for regulating cell osmotic pressure.

5. Use of the compound ABAGG according to claim 1 or 2 as a substance for improving the salt tolerance of organisms.

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