Streptomyces HU154 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a Streptomyces sp.HU154 and application thereof, the strain is named Streptomyces sp.HU154, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.28456, the strain comes from a farmland soil sample in the Hengqiao town in Changxian County, Huzhou City, Zhejiang Province, and the strain can be used for fermentation production of dequasol. After the strain is subjected to aerobic liquid submerged fermentation, the content of the diquathrin in fermentation liquor can reach 3850ug / ml, and the yield of the diquathrin produced by fermentation is high.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Streptomyces HU154 and its application. Background Art

[0002] Decumycin, also known as Angustmycin A, PRG-08 or Guvermectin, is a nucleoside compound isolated from Streptomyces hygroscopics var. angustmyceticus by Yuntsen et al. in 1958. Since its discovery in the 1950s, it has not attracted much attention due to its extremely narrow antibacterial spectrum and weak antibacterial activity. Scientific workers have found that it has the biological activity of plant cytokinin, which has attracted extensive attention from scientific researchers. This compound has been reported to promote wheat seed germination and seedling growth, promote the drought resistance of wheat seedlings, induce the differentiation of potato callus, significantly promote the occurrence and proliferation of Panax notoginseng callus, promote the occurrence and seedling formation of Panax notoginseng embryoids; it can induce the preferential germination and rapid growth of axillary buds of explants in the tissue culture of Siraitia grosvenorii, promote the preservation of the vegetable Chrysanthemum nankingense, etc., showing good application prospects and social value in botany and agronomy.

[0003] However, the currently disclosed decumycin-producing strains are Streptomyces NEAU6 and angustmyceticus disclosed in CN109694838A, and S. hygroscopicus subsp. angustmyceticus NRRLB-2347 disclosed by Yashawant K et al. However, the fermentation levels of decumycin produced by the above two strains are relatively low, which affects the industrial development of this compound. Therefore, if high-yield strains can be obtained, it is of great significance for the industrialization of decumycin. Summary of the Invention

[0004] The present invention provides a strain of Streptomyces, named Streptomyces sp. HU154. This strain is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO. 28456. This strain can ferment and produce decumycin, and the content of decumycin in the fermentation broth can reach 3850 μg / ml after aerobic liquid deep fermentation.

[0005] The specific technical solution of the present invention is as follows: A strain of Streptomyces, named Streptomyces sp. HU154; it is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 28456.

[0006] The present invention provides a strain of Streptomyces HU154, the source of which is a farmland soil sample from Hongqiao Town, Changxing County, Huzhou City, Zhejiang Province; this strain is named Streptomyces sp. HU154; this strain can be used to prepare dequalinium; this strain is currently deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO. 28456, and the deposit date is September 13, 2023. The address of the depositary institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] An application of the above Streptomyces HU154 in the preparation of dequalinium, comprising the following steps: (1) Inoculate Streptomyces HU154 into a seed medium for seed culture to prepare a seed solution; (2) Inoculate the seed solution into a fermentation medium for fermentation culture to obtain a fermentation broth; (3) Purify dequalinium in the fermentation broth to obtain a dequalinium product.

[0008] The present invention also provides an application of the above Streptomyces HU154 in the preparation of dequalinium. After aerobic liquid submerged fermentation using Streptomyces sp. HU154, 3850 μg / ml of dequalinium can be produced in the fermentation broth, and the yield of dequalinium is high.

[0009] Preferably, the raw materials of the seed medium in step (1) include glucose, sucrose, hot-exploded soybean cake powder, cottonseed cake powder, and calcium carbonate.

[0010] Preferably, the conditions for seed culture in step (1) include: culturing on a shaker at room temperature, the shaker speed is 100 - 300 rpm, and the fermentation time is 40 - 48 h.

[0011] Preferably, the pH of the seed medium is 6.8 - 7.5.

[0012] Preferably, the raw materials of the fermentation medium in step (2) include: corn soluble starch, glucose, hot-exploded soybean cake powder, cottonseed cake powder, light calcium carbonate, sodium chloride, and magnesium sulfate heptahydrate.

[0013] Preferably, the conditions for fermentation culture in step (2) include: temperature 25 - 33 °C, pH 4.0 - 8.0, aeration rate 0.5 - 2.0 vvm, and fermentation time 120 - 240 h.

[0014] Preferably, the purification step in step (3) is as follows: centrifuging the fermentation broth to obtain the supernatant, separating the supernatant through macroporous resin and silica gel column chromatography to obtain the crude product, and recrystallizing the crude product to obtain the decarboxylase product.

[0015] Preferably, the macroporous resin separation uses an HP-816 resin column for separation, and the elution of the macroporous resin separation is carried out with ethanol.

[0016] Preferably, the elution of the silica gel column chromatography separation is carried out using a chloroform / methanol gradient elution.

[0017] Compared with the prior art, the present application has the following technical effects: (1) The present invention provides a strain of Streptomyces, the original source of which is isolated from a farmland soil sample in Hongqiao Town, Changxing County, Huzhou City, Zhejiang Province; this strain is named Streptomyces sp. HU154; this strain can be used to prepare decarboxylase; this strain is currently deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO. 28456, and the deposit date is September 13, 2023; (2) The present invention also provides the application of Streptomyces HU154 in the preparation of decarboxylase. After aerobic liquid deep fermentation using Streptomyces sp. HU154, the content of decarboxylase in the fermentation broth can reach 3850 μg / ml, and the yield of decarboxylase is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the SEM image of the mycelial morphology of Streptomyces sp. HU154 of the present invention.

[0019] Figure 2 It is the colony morphology image of Streptomyces sp. HU154 of the present invention on different culture media.

[0020] Figure 3 It is the HPLC chromatogram of decarboxylase and decarboxylase standard product in the fermentation broth prepared in Example 5 of the present invention.

[0021] Figure 4 It is the HRESIMS chromatogram of the decarboxylase product prepared in Example 6 of the present invention.

[0022] Figure 5 It is the 1 1H-NMR chromatogram of the decarboxylase product prepared in Example 6 of the present invention.

[0023] Figure 6 It is the 13 13C-NMR chromatogram of the decarboxylase product prepared in Example 6 of the present invention.

[0024] Figure 7 This is the DEPT135 spectrum of the decarbasugramycin product obtained in Example 6 of the present invention. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with the embodiments.

[0026] Example 1: Isolation of the strain Streptomyces sp. HU154 (CGMCC NO. 28456) The Streptomyces sp. HU154 was isolated from a farmland soil sample in Tuying Village, Hongqiao Town, Changxing County, Huzhou City, Zhejiang Province.

[0027] A total of 8 soil samples were collected from the Tuying Wetland by the Taihu Lake. The samples were soils with a depth of 5 - 15 cm, and each was about 100 g. After mixing the obtained soil samples, 20 g was taken and ground finely in a sterile mortar, then added to a conical flask containing 150 ml of sterile water, and 150 μl of nalidixic acid (concentration: 50 mg / ml) was added. It was oscillated at room temperature for 1 h (150 rpm) to form a suspension with a concentration of 10 0 . Then the 10 0 suspension was serially diluted with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5The concentration was taken, and 0.15 ml of each concentration of the dilution was spread on the Gause's No. 1 medium and the humic acid-vitamin (HV) medium plate medium added with nalidixic acid (final concentration of 50 μg / ml) and cycloheximide (final concentration of 30 μg / ml). Two parallel groups were spread for each group of samples. The two parallel groups were placed in a constant temperature incubator at 28°C for cultivation. On the 4th day and the 7th day respectively, actinomycete colonies were picked, and each picked strain was spot-inoculated on two kinds of media, namely ISP2 plate and Gause's No. 1 plate, and cultured at 28°C for 7 days, and then purified. More than 140 actinomycetes were obtained in total. The obtained strains were cultured on YMS (0.4% yeast extract, 1.0% malt extract, 1.0% soluble starch, 1.8% agar, pH 7.0) plate, and then the colonies were scraped off with an inoculation shovel under sterile conditions and inoculated into a 250-ml conical flask, with 30 ml of seed medium in each flask. The culture was shaken at 28°C and 240 rpm for 48 h to obtain the seed liquid, and then the seed liquid was inoculated into different 250-ml conical flasks containing fermentation medium at an inoculation amount of 5-10%, with 30 ml of fermentation medium in each flask. The culture was shaken at 28°C and 240 rpm for 7 days; 3 volumes of methanol were added to the fermentation broth, and ultrasonic treatment was carried out for 30 min, and then filtered with filter paper. The content of dequalinium in the obtained fermentation broth was detected by HPLC. The strains with the same peak time as the standard dequalinium were selected, and then through LC-MS detection, it was confirmed that the compound peak with the same liquid phase retention time as the standard product in the fermentation product was the dequalinium peak. Finally, a strain with high yield of dequalinium, namely Streptomyces sp. HU154, was obtained.

[0028] Example 2: The morphological characteristics of Streptomyces sp. HU154 (CGMCC NO. 28456) were experimented according to the methods disclosed in "Streptomyces Identification Manual", "Classification and Identification of Actinomycetes" and "Common Bacterial System Identification Manual". The judgment of color was carried out with reference to the colors in the RALK7 color card.

[0029] The mycelial morphology of Streptomyces sp. HU154 was observed by scanning electron microscope (SEM), and the results are shown in Figure 1 , as Figure 1 shown, it can be seen that the mycelia are coral-shaped and curved.

[0030] Strain culture characteristics: Streptomyces sp. HU154 was cultured on a flat plate for 10 days using 7 media recommended by the International Streptomyces Projects Medium (ISP1 medium, ISP2 medium, ISP3 medium, ISP4 medium, ISP5 medium, ISP6 medium, and ISP7 medium), Bennett medium, nutrient agar medium, Czapek medium, Gao's No. 1 medium, and starch medium; the colony morphology of Streptomyces sp. HU154 was observed, and the results of the morphological characteristics are shown in Figure 2 .

[0031] As Figure 2 shown, the colony morphology of Streptomyces sp. HU154 on the above different media is as follows: (1) On ISP1 medium, the colony diameter is 12 mm, the colony morphology is irregularly round, the edge is wavy, the surface has a small amount of folds, the colony surface is off-white, the substrate mycelium is sandy yellow, and spore production is abundant; (2) On ISP2 medium, the colony diameter is 13 mm, the colony morphology is irregularly round, the edge is serrated, the surface has no folds, the colony surface is grayish white, the edge is gray, the substrate mycelium is off-white, and spore production is abundant; (3) On ISP3 medium, the colony diameter is 15 mm, the colony morphology is irregularly round, there are horizontal cracks at the edge, the color of the colony edge is light and the middle color is deep, with gray spores, and spore production is very abundant; (4) On ISP4 medium, the colony diameter is 11 mm, the colony morphology is irregularly round, the edge is wavy, there is a transparent halo at the edge, the color of the colony surface is light at the edge and deep in the middle, the substrate mycelium is light gray, and spore production is very abundant; (5) On ISP5 medium, the colony diameter is 12 mm, the colony morphology is irregularly round, the edge is wavy, the colony has many folds, the colony surface is light yellow, the substrate mycelium is light yellow, and spore production is abundant; (6) On ISP6 medium, the colony diameter is 10 mm, the colony morphology is irregularly round, the edge is wavy, the surface is smooth, the colony surface is yellowish brown, the substrate mycelium is light brown, and there are no spores; (7) On ISP7 medium, the colony diameter is 9 mm, the colony morphology has a wavy edge, the colony has many folds and irregular cracks, the colony surface is off-white, the substrate mycelium is light brown, and spore production is abundant; (8) On Bennett medium, the colony diameter is 11 mm, the colony morphology is irregularly round, with radioactive folds on the surface, the edge is serrated, the colony surface is light gray, the substrate mycelium is light yellow, and spore production is abundant; (9) The colony diameter is 13 mm on nutrient agar medium. The colony morphology shows irregular cracks in the middle with relatively deep cracks, the edge is irregular and serrated, the colony surface is light yellow, the substrate mycelium is light yellow, and spore production is abundant; (10) The colony diameter is 11 mm on Czapek's medium. The colony morphology is irregularly round, the surface is relatively flat, the edge is smooth and regular, the colony surface is gray, the substrate mycelium is light yellow, and spore production is abundant; (11) The colony diameter is 10 mm on Gao's No. 1 medium. The colony morphology is irregularly round, the surface is not smooth, there are irregular cracks in the middle, the edge is wavy, the colony surface is white, the substrate colony is sandy yellow, and spore production is abundant; (12) The colony diameter is 13 mm on starch medium. The colony morphology is irregularly round, the spores do not completely cover the colony, there is a hydrolytic transparent circle, the edge is smooth and there are no spores, the colony surface is off-white, the substrate colony is sandy yellow, and spore production is abundant.

[0032] Example 3: Physiological and biochemical characteristics of Streptomyces sp. HU154 (CGMCC NO. 28456) (1) Carbon source utilization experiment: Add 2.64 g of ammonium sulfate, 2.64 g of potassium hydrogen phosphate, 5.65 g of dipotassium hydrogen phosphate, 1 g of magnesium sulfate heptahydrate, 0.0064 g of copper sulfate pentahydrate, 0.0011 g of ferric sulfate heptahydrate, 0.0079 g of manganese chloride tetrahydrate, and 0.0015 g of zinc sulfate heptahydrate to 1000 ml of distilled water and adjust the pH to 6.5. Then, prepare media using sucrose, galactose, trehalose, glucose, inositol, sorbitol, mannitol, raffinose, xylose, lactose, xylan, rhamnose, fructose, ribose, arabinose, and maltose respectively, and set up a blank medium without adding a carbon source. Inoculate Streptomyces sp. HU154 into the above media and culture at 28 °C for 10 days. The culture results are shown in Table 1; (2) Nitrogen source utilization experiment: Add 1 g of D-glucose, 0.05 g of magnesium sulfate heptahydrate, 0.05 g of sodium chloride, 0.001 g of ferric sulfate heptahydrate, and 0.01 g of dipotassium hydrogen phosphate to 100 ml of water and adjust the pH to 7.2. Then, prepare media using 0.5 g of glycine + 1.8 g of agar, 0.5 g of tyrosine + 1.8 g of agar, and 0.5 g of asparagine + 1.8 g of agar respectively, and set up a blank medium without adding a nitrogen source. Inoculate Streptomyces sp. HU154 into the above media and culture at 28 °C for 10 days. The culture results are shown in Table 1; Table 1 Carbon source and nitrogen source utilization experiments Carbon source Growth condition Nitrogen source Growth condition Sucrose 4 Tyrosine 3 Galactose 3 Serine 4 Trehalose 3 Threonine 4 Glucose 2 Alanine 4 Inositol 4 Glutamine 3 Sorbitol 4 Proline 3 Mannitol 3 Creatine 4 Raffinose 4 Asparagine 2 Xylose 1 Glycine 3 Lactose 2 Arginine 4 Xylan 2 / / Rhamnose 2 / / Fructose 0 / / Ribose 0 / / Arabinose 0 / / Maltose 4 / / As shown in Table 1, the meanings of the numbers in Table 1 are as follows: 0, no growth; 1, very weak growth; 2, can grow, with a small amount of spores; 3, good growth, with a large amount of spores; 4, best growth, with abundant spores. It can be seen from the results in Table 1 that the preference order of Streptomyces sp. HU154 for carbon sources is: the best are sucrose, inositol, sorbitol and raffinose; followed by galactose, trehalose and mannitol; then glucose, lactose, xylan and rhamnose; the worst is xylose; fructose, ribose and arabinose cannot be utilized. The preference order of Streptomyces sp. HU154 for nitrogen sources is: the best are serine, threonine, alanine, creatine and arginine; followed by tyrosine, glutamine and glycine; then aspartic acid.

[0033] (3) Physiological and biochemical characteristic tests: The physiological and biochemical characteristics of Streptomyces sp. HU154 were tested by the methods disclosed in the Manual for Systematic Identification of Common Bacteria. The test items included gelatin liquefaction experiment, starch hydrolysis experiment, milk coagulation experiment, esterase Tween 40 experiment, aesculin experiment, nitrate reduction experiment, hydrogen sulfide production experiment, esterase Tween 60 experiment, cellulose utilization experiment, catalase experiment, urease experiment and esterase Tween 80 experiment. The test results are shown in Table 2. Table 2 Physiological and biochemical characteristics Item Result Gelatin liquefaction - Starch hydrolysis + Milk coagulation + V.P. test - Esculin + Nitrate reduction - Hydrogen sulfide production + Lipase - Urease + As shown in Table 2, in Table 2, "+" represents positive and "-" represents negative. It can be seen from the results in Table 2 that Streptomyces sp. HU154 is positive for tests such as starch hydrolysis, milk coagulation, aesculin, hydrogen sulfide production and urease, and Streptomyces sp. HU154 is negative for tests such as gelatin liquefaction, V.P. experiment, nitrate reduction and esterase.

[0034] (4) Using ISP medium as the basal medium, test media with different NaCl concentrations, different pH values and different temperatures were prepared respectively to culture Streptomyces sp. HU154 for 10 days, and the growth conditions of Streptomyces sp. HU154 were observed. The results are shown in Table 3. Table 3 Colony growth conditions at different NaCl concentrations, different pH values and different temperatures As shown in Table 3, "0" in Table 3 indicates good growth; "1" indicates relatively weak growth; "2" indicates weak growth with a small amount of spores; "3" indicates good growth with a large amount of spores; "4" indicates vigorous growth with abundant spores. It can be seen from the results in Table 3 that Streptomyces sp. HU154 grows best on ISP medium at pH 7 - 8, grows well on ISP medium at pH 5 - 6 and 9, and grows poorly at other pH values; Streptomyces sp. HU154 grows best on ISP medium at a temperature of 20 - 28 °C, grows well on ISP medium at 16 °C, and grows poorly at other temperatures; Streptomyces sp. HU154 grows best on ISP medium at an NaCl concentration of 0%, grows well on ISP medium at an NaCl concentration of 1 - 5%, and grows poorly at other NaCl concentrations.

[0035] Example 4: 16S rDNA sequence analysis and strain identification of Streptomyces sp. HU154 (1) 16S rDNA sequence analysis of strain HU154

[0036] Table 4 Homology between Streptomyces sp. HU154 and type strains As shown in Table 4, it can be seen from the results of Table 4 that the 16S rDNA sequence of Streptomyces sp. HU154 has a high homology with Streptomyces sp., up to more than 99.5%. Combining the above morphological, cultural characteristics and physiological and biochemical characteristics, the strain Streptomyces sp. HU54 was identified as Streptomyces sp.

[0037] (2) The comparison between strain HU154 and the reported decumycin-producing strains is as follows: The strain NEAU6 (CGMCC 16338) reported in Patent CN 109694838 A can produce wugufengsu, and the structure of wugufengsu is the same as that of decumycin. The titer of such compounds produced by it was not mentioned in the article. The strain reported by Liu C X et al. (Liu CX, Bai L, Cao P, et al. Journal of Agricultural and Food Chemistry, 2022, 70(51): 6229-16240) is the same strain as the strain reported in Patent CN 109694838 A. The decumycin yield of strain HU154 can reach more than 3600 μg / ml. The strain was cultured on ISP3 medium for two weeks. The aerial mycelium was gray and the substrate mycelium was brownish black. The edge of the colony of strain HU154 was light gray and the middle was dark gray, and the substrate mycelium was off-white. Under scanning electron microscopy, the aerial mycelium of Streptomyces NEAU6 was spiral, while the mycelium of strain HU154 was partially curved and coral-like, showing obvious differences. In addition, the similarity was 99.57% when the 16S rDNA sequences (GenBank No. KX679573.2) of strain HU154 and Streptomyces NEAU6 were aligned, showing great differences.

[0038] The angustmycin-producing strain Streptomyces hygroscopicus subsp. angustmyceticus reported by Yuntsen H et al. (Yuntsen H, Ohkuma K, Ishii Y, et al. J Antibiot, 1956, 9(6):195) is the same strain as the strain S. hygroscopicus subsp. angustmyceticus NRRL B-2347 reported by Yashawant K et al. (Yashawant K, Michael G, Int J Syst Evol Micr, 2010, 60:769–775). Its physiological and biochemical characteristics are as follows: aerial mycelium gradually changes from gray to black and is moist, substrate mycelium is dark yellow, and there is no soluble pigment; the available carbon sources (1% w / v) include D-arabinose, cellobiose, dextrin, D-fructose, L-fucose, D-galactose, glycogen, inositol, melezitose, D-ribose, D-xylose, 1,4-butanediol; it cannot utilize ribose, L-arabinose, erythritol, α-lactose, L-rhamnose, raffinose, salicin, D-sorbitol, and cannot hydrolyze starch; the available nitrogen sources (1% w / v) include L-glutamic acid, L-leucine, L-proline; it cannot grow at pH 5.0 and below, but can grow at pH 10.0; it cannot grow at 10°C and below, but can grow at 37°C. The differences in physiological and biochemical characteristics between strain HU154 and it are as follows: it can utilize α-lactose, L-rhamnose, raffinose, D-sorbitol, and can hydrolyze starch; it cannot grow at 37°C; in addition, the similarity between the reported strain S. hygroscopicus subsp. angustmyceticus NRRL B-2347 and its type strain (DSM 41683 T / NRRL B-2347 T ) and strain HU154 is only 99.14%, with a relatively large difference.

[0039] The decoyinine-producing strain Streptomyces hygroscopicus subsp. decoyicus reported by Vavra, J J, et al. (Vavra J J, Dietz A, Churchill B W, et al. Antibiot Chemother, 1959, 9: 427–431) and the strain S. hygroscopicus subsp. angustmyceticus NRRL B-2347 reported by Yashawant K, et al. (Yashawant K, Michael G, Int J Syst Evol Micr, 2010, 60: 769–775) are the same strain. Its physiological and biochemical characteristics are as follows: aerial mycelium is white, substrate mycelium is dark yellow, and there is no soluble pigment; the available carbon sources (1% w / v) include D-arabinose, cellobiose, dextrin, D-fructose, raffinose, D-galactose, glycogen, melezitose, D-sorbitol, 1,4-butanediol; ribose, L-arabinose, erythritol, L-fucose, inositol, α-lactose, L-rhamnose, D-ribose, salicin, D-xylose cannot be utilized, and starch cannot be hydrolyzed; the available nitrogen sources (1% w / v) include L-glutamic acid, L-leucine, L-proline; it cannot grow at pH 5.0 and below, but can grow at pH 10.0; it can grow at 10 °C and 37 °C. The differences in physiological and biochemical characteristics between strain HU154 and it are as follows: it can utilize L-fucose, inositol, α-lactose, L-rhamnose, D-xylose, and can hydrolyze starch; it cannot grow at 37 °C; in addition, the type strain (DSM 41427 T / 5NRRL 2666 T ) of the reported strain S. hygroscopicus subsp. angustmyceticus NRRL B-2347 has only 99.14% similarity with strain HU154, showing a relatively large difference.

[0040] The angustmycin A-producing strain reported by Yu L, et al. (Yu L, Zhou W, She Y, et al. Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system. Nat Commun, 2021, 12, 6633.) is the same strain as Streptomyces decoyicus NRRL 2666.

[0041] In summary, considering other characteristics of Streptomyces sp. HU154 (CGMCC NO. 28456) in the present invention described above, it can be known that strain HU154 belongs to the genus Streptomyces, but it is different from the known decarboxylase producers (Streptomyces sp. NEAU6, S. hygroscopicus subsp. angustmyceticus NRRL B-2347, CCTCC M90003). Therefore, Streptomyces sp. HU154 (CGMCC NO. 28456) is a new decarboxylase-producing strain.

[0042] Example 5: Preparation of a fermentation broth containing decarboxylase compound using Streptomyces sp. HU154 (1) Inoculate Streptomyces sp. HU154 onto a YMS plate medium (10 g of soluble starch, 4 g of yeast extract, 10 g of malt extract, made up to 1000 ml with purified water, pH 7.0, 18 g of agar, sterilized at 121 °C for 20 min, poured into plates when cooled to about 55 °C,) and culture at 28 °C for 10 days; (2) Scrape the colony spores on the plate in step (1) and inoculate them into a seed medium (10 g of glucose, 10 g of sucrose, 15 g of hot-fried soybean cake powder, 15 g of cottonseed cake powder, 2 g of CaCO3, 1000 ml of purified water, pH 7.0 before sterilization, filled in a 250-ml Erlenmeyer flask with 25 ml, sterilized at 121 °C for 20 min) and perform shaking culture under the conditions of a temperature of 25 °C, a rotation speed of 200 rpm, and a culture time of 48 hours until the pH of the seed liquid reaches 7.0 and the mycelium concentration is 20-30% to complete the seed liquid culture; (3) Inoculate the seed liquid into a fermentation medium (60 g of corn soluble starch, 20 g of glucose, 20 g of hot-fried soybean cake powder, 20 g of cottonseed cake powder, 2 g of light calcium carbonate, 2 g of sodium chloride, 2 g of magnesium sulfate heptahydrate, 1000 ml of purified water, pH 6.0 before sterilization, filled in a 250-ml Erlenmeyer flask with 25 ml, sterilized at 121 °C for 20 min) at an inoculation amount of 10% by volume, then place it in a shaker and shake culture at 25 °C and 200 rpm for 200-240 h. After the fermentation is completed, use HPLC to detect the content of decarboxylase in the fermentation broth, and the content of decarboxylase is 3850 μg / ml.

[0043] Example 6: Preparation of a decarboxylase product using Streptomyces sp. HU154 (1) Inoculate Streptomyces sp. HU154 onto a YMS plate medium (10 g of soluble starch, 4 g of yeast extract, 10 g of malt extract, make up to 1000 ml with purified water, pH 7.0, 18 g of agar, sterilize at 121 °C for 20 min, pour into plates when cooled to about 55 °C), and culture at 28 °C for 10 days; (2) Scrape the colony spores on the plate in step (1) and inoculate them into a seed medium (10 g of glucose, 10 g of sucrose, 15 g of hot-exploded soybean cake powder, 15 g of cottonseed cake powder, 2 g of CaCO3, 1000 ml of purified water, pH 7.0 before sterilization, filled in a triangular flask at 25 ml / 250 ml, sterilize at 121 °C for 20 min), and perform shake flask culture at a temperature of 25 °C, a rotation speed of 200 rpm for 40 - 48 h until the pH of the seed liquid is 7.0 and the mycelium concentration is 20 - 30% to complete the first-stage seed liquid; (3) Add 8 L of the seed medium in step (2) to a 15 L seed tank, sterilize with high-pressure steam at 121 °C for 30 min, after cooling to 30 °C, inoculate 5 L of the first-stage seed liquid, stir at a rotation speed of 150 - 500 rpm (adjust the rotation speed according to dissolved oxygen, control the dissolved oxygen not less than 30%), the ventilation rate is 1 - 3 vvm (adjust the rotation speed according to dissolved oxygen, control the dissolved oxygen not less than 30%), perform deep fermentation at 28 °C for 220 h, end the fermentation and empty the tank, use the HPLC method to detect the content of dequalinium in the fermentation broth, and the content of dequalinium is 3680 μg / ml; (4) Take 30 L of the fermentation broth in step (3), centrifuge at 4000 rpm for 15 min to obtain the supernatant. After passing the supernatant through an HP-816 resin column, first wash with 1 - 2 volumes of water, then elute with ethanol (95%) for 1 - 2 column volumes, collect the ethanol elution part and concentrate it under reduced pressure to obtain an extract containing dequalinium. After mixing the extract and silica gel evenly, load it onto a silica gel column, use a chloroform / methanol gradient elution with a volume ratio of 90:10 - 50:50, collect the elution parts in segments, perform TLC detection (volume ratio of chloroform / methanol = 9:1), combine the fractions with a chromatographic purity greater than 90%, and vacuum concentrate to dryness to obtain the crude dequalinium. The crude dequalinium is recrystallized with water to obtain 45.4 g of the dequalinium product.

[0044] Detection example: Perform HPLC testing on the dequalinium in the fermentation broth of Example 5, and the HPLC chromatogram of the test is shown in Figure 3 ; Perform testing on the dequalinium product prepared in Example 6, and the test items include HRESIMS, 1H NMR, 13 13C NMR and DEPT135 and nuclear magnetic resonance, and the HRESIMS spectrum is shown in Figure 4 ,1 The 1H NMR spectrum is shown in Figure 5 , 13 the 13C NMR spectrum is shown in Figure 6 and the DEPT135 spectrum is shown in Figure 7 , and the NMR results are shown in Table 5; Table 5 NMR data of decumycin (DMSO-d6) As Figure 4 , Figure 5 , Figure 6 , Figure 7 and as shown in Table 5, the pure decumycin obtained in Example 6 was analyzed by HRESIMS (m / z 280.1048 [M+H] + ) and its molecular weight was 279, and the chemical formula was calculated as C 11 H 13 N5O. According to the content reported in the literature (Yu, L., Zhou, W., She, Y. et al. Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system. Nat Commun, 2021, 12, 6633.), it was determined that the purified product of the fermentation broth obtained in the present invention was decumycin.

[0045] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A Streptomyces sp. HU154, characterized in that, The strain is named Streptomyces sp. HU514; it is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 28456.

2. Use of Streptomyces sp. HU154 according to claim 1 in the preparation of decumycin, characterized in that, It includes the following steps: (1) Inoculate Streptomyces HU154 into a seed medium for seed culture to prepare a seed solution. (2) Inoculate the seed solution into a fermentation medium for fermentation culture to obtain a fermentation broth. (3) Purify decadensin in the fermentation broth to obtain a decadensin product.

3. The application according to claim 2, characterized in that, The raw materials of the seed medium in step (1) include glucose, sucrose, hot-fried soybean cake powder, cottonseed cake powder, and calcium carbonate.

4. The application according to claim 2 or 3, characterized in that, The conditions for seed culture in step (1) include: culturing on a shaker at room temperature, with a shaker speed of 100 - 300 rpm and a fermentation time of 40 - 48 h.

5. The application according to claim 3, characterized in that, The pH of the seed medium is 6.8 - 7.

5.

6. The application according to claim 2, characterized in that The raw materials of the fermentation medium in step (2) include: corn soluble starch, glucose, hot-fried soybean cake powder, cottonseed cake powder, light calcium carbonate, sodium chloride, and magnesium sulfate heptahydrate.

7. The application according to claim 2 or 6, characterized in that, The conditions for fermentation culture in step (2) include: a temperature of 25 - 33 °C, a pH of 4.0 - 8.0, an aeration rate of 0.5 - 2.0 vvm, and a fermentation time of 120 - 240 h.

8. The application according to claim 2, characterized in that, The purification steps in step (3) are: centrifuge the fermentation broth to obtain the supernatant, separate the supernatant through a macroporous resin column and a silica gel chromatography column to obtain a crude product, and recrystallize the crude product to obtain a decadensin product.

9. The application according to claim 8, wherein The macroporous resin separation uses an HP-816 resin column for separation, and the elution of the macroporous resin separation is carried out with ethanol.

10. The application according to claim 8, characterized in that, The elution of the silica gel column chromatography separation uses a chloroform / methanol gradient elution.

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