CN112746036A

CN112746036A - Streptomyces and method for producing pseudouridine by fermenting same

Info

Publication number: CN112746036A
Application number: CN202011421953.7A
Authority: CN
Inventors: 陈伟; 郑玲辉; 朱进伟; 高祥; 张敏; 陈世敏; 彭湘屏; 汪超
Original assignee: Zhejiang Hunda Biotechnology Co ltd
Current assignee: Zhejiang Hunda Biotechnology Co ltd
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-05-04
Anticipated expiration: 2040-12-08
Also published as: CN112746036B

Abstract

The invention discloses a Streptomyces sp HDCC00030 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.21122 and the preservation date of 2020, 11 and 6 days. The streptomycete is a brand-new pseudouridine producing strain, has high production capacity, has the capability of producing pseudouridine greatly improved compared with other strains in the prior art, has the titer of the pseudouridine reaching over 1360mg/L, and is beneficial to realizing industrial production.

Description

Streptomyces and method for producing pseudouridine by fermenting same

Technical Field

The invention relates to the technical field of industrial microbial fermentation, in particular to streptomyces and a method for producing pseudouridine by fermenting the streptomyces.

Background

In 1957, DAVIS et al first discovered an isoform similar to the natural structure of Uridine in ribosomal RNA (ribosomal RNA, rRNA) and transfer RNA (transfer RNA, tRNA), whose ribose was not linked to uracil (U) N1, but to C5 to form Pseudouridine (PU). (DAVISFF, ALLEN F W. Ribonucleic acids from yellow last white conjugate a F-th nucleotide [ J ]. JBiol Chem, 1957, 227 (2): 907-. The chemical structural formulas of the uridine and the pseudouridine are shown as a formula 1 and a formula 2;

pseudouridine is catalyzed by a pseudouridine synthase, which isomerizes specific uridines in RNA after transcription, a process called pseudouridine. Pseudouridine is a nucleotide metabolite, and studies on it have been increasingly focused on by researchers. The pseudouridine can be used as a potential biomarker for diagnosing and monitoring the curative effect of nephropathy and tumors, and has certain reference significance for diagnosing other diseases. Studies have shown that β -pseudoouridine can reduce radiation-induced chromosomal aberrations in human lymphocytes. In recent years, scientists speculated that pseudouridine plays a role in RNA stability and/or assisting aminoacylase interactions with tRNAs. In addition, with the rapid development of biotechnology, genomic drugs discovered by means of aptamers, RNA interference, and the like have attracted interest, and pseudouridine is used as a pharmaceutical intermediate and a phosphoramidine derivative of pseudouridine is used as one of starting materials for synthesizing such oligomers, according to its specific physiological function.

In the preparation of pseudouridine, currently, only the traditional chemical synthesis method can catalyze and synthesize pseudouridine, but the chemical synthesis of pseudouridine has a series of problems of long chemical synthesis steps, low yield, flammable and explosive reagents, low safety and the like. In the aspect of biosynthesis, no microbial strain and technical report which can be exclusively used for preparing pseudouridine in a large amount are found. Thus, the low cost convenient acquisition of pseudouridine has certain drawbacks.

Aiming at the problems that the pseudouridine can only be prepared by a chemical synthesis method in the prior art, the chemical synthesis process has long synthesis steps and low yield, and used reagents are inflammable, explosive and unsafe, the invention seeks a novel microorganism and can realize the production of the pseudouridine by simple fermentation and low cost.

Disclosure of Invention

In order to solve the problem of insufficient existing pseudouridine preparation methods, one of the purposes of the invention is to provide a novel pseudouridine-producing strain, which is Streptomyces sp (HDCC) 00030 and is preserved in China general microbiological culture Collection center (CGMCC), Beijing, China, with the preservation number of CGMCC NO.21122 and the preservation date of 2020, 11 months and 6 days.

The invention also aims to provide application of Streptomyces sp HDCC00030(CGMCC NO.21122) in preparing pseudouridine or a pharmaceutical composition containing the pseudouridine.

The invention also provides a preparation method of pseudouridine, which comprises the step of carrying out aerobic fermentation by adopting Streptomyces sp HDCC00030(CGMCC NO.21122) in a nutrient medium containing assimilable carbon sources and/or nitrogen sources.

In a preferred embodiment, the assimilable carbon source is selected from one of corn starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or a combination of any of the above, preferably corn starch, maltodextrin, glucose, sucrose, sorbitol or a combination of any of the above.

In a preferred embodiment, the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salt or a combination of any of the above substances, preferably soybean cake powder, yeast extract powder, corn steep liquor dry powder, yeast powder or a combination of any of the above substances.

In a preferred embodiment, the nutrient medium further comprises an inorganic salt selected from one of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate or a combination of any of the above substances, preferably calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride or a combination of any of the above substances.

In a preferred embodiment, the nutrient medium contains 5-50g/L of corn starch, 5-50g/L of maltodextrin, 5-60g/L of glucose, 5-50g/L of sorbitol, 1-15g/L of yeast extract powder, 5-20g/L of soybean cake powder, 5-20g/L of corn steep liquor dry powder, 1-10g/L of magnesium sulfate, 2-8g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium chloride and 3-50g/L of calcium carbonate.

In a preferred embodiment, the temperature of the aerobic fermentation is 20-35 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24-240 hours, preferably 72-168 hours; the oxygen throughput is from 0.1 to 2.0vvm, preferably from 0.5 to 2.0 vvm.

In a preferred embodiment, said Streptomyces sp HDCC00030 is said fermentatively cultured by seed liquid inoculation into said nutrient medium; the seed solution is obtained by seed culture of streptomyces HDCC00030 in a seed culture medium.

In a preferred embodiment, the seed culture medium contains 5-30g/L of glucose, 5-20g/L of corn starch, 2-10g/L of soybean cake powder, 1-10g/L of yeast extract powder, 1-20g/L of calcium carbonate, 1-10g/L of magnesium sulfate and 1-10g/L of dipotassium phosphate.

In a preferred embodiment, the seed culture conditions are: the temperature of seed culture is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24 to 80 hours, preferably 24 to 60 hours.

The pseudouridine of the present invention was detected by HPLC under the following conditions:

the liquid phase detection method conditions used for the pseudouridine titer detection are as follows:

a chromatographic column: waters Xbridge Amide (250 mm. times.460 mm,3.5 μm), mobile phase A: 0.2% (mass to volume, unit is g/L) of ammonium acetate aqueous solution, mobile phase B: acetonitrile, retention time: 20min, flow rate: 1mL/min, sample size: 10 μ L, detection wavelength: 255nm, column temperature: (25. + -. 1). degree.C.

TABLE 1 elution procedure volume ratio of mobile phases A and B

Min	A	B
			0	100	0
3	100	900
			6	98	2
12	60	40
			17	100	0
20	100	0

The Streptomyces sp HDCC00030 has the following main biological characteristics: the colony is oval in shape, multiple folds and bulges are formed on the surface, the diameter of the colony is about 8-20 mm, furrows are formed in the surface of the colony, the middle of the colony is slightly concave, matrix hypha is developed, the colony is tightly combined with a culture medium and is not easy to pick up, the color is beige or faint yellow, aerial hypha is white, sporulation is rich, the early stage is white, the later stage is changed into light yellow, and no soluble pigment exists.

The bacterial strain (Streptomyces sp.) HDCC00030 is a brand-new pseudouridine producing strain, has high production capacity, has greatly improved pseudouridine producing capacity compared with other strains in the prior art, has the titer of the pseudouridine reaching over 1360mg/L, and is beneficial to realizing industrial production.

Drawings

FIG. 1 is a micrograph (400X) of strain HDCC00030 on ISP2 medium.

FIG. 2 is a characteristic map of the colonies of strain HDCC00030 on ISP2 medium.

FIG. 3 shows the pseudouridine chromatogram obtained by separating and extracting the bacterial cells after fermentation culture of the bacterial strain HDCC00030 and detecting the pseudouridine by HPLC.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials, reagents and the like used in the following examples are all common commercially available products and are commercially available unless otherwise specified.

Wherein the actinomycete DNA extraction kit is purchased from Beijing Sanbo Polygala tenuifolia biotechnology, LLC;

purification and recovery of PCR products the SanPrep column type PCR product purification kit used was purchased from Biotechnology engineering (Shanghai) Ltd.

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope.

Example 1: the source of the strain

Streptomyces sp HDCC00030 is separated from soil of mountain slopes of the urban Qingcheng province in Sichuan of China.

Cross sampling is carried out on soil in the Qingcheng mountain area, 5 sampling points are randomly selected, 10g of soil sample is taken at each point, the soil samples are put into a conical flask, 10g of samples are taken after uniform mixing, the samples are added into the conical flask filled with 90mL of sterile water (a magnetic stirrer is arranged in the flask), vortex stirring is carried out for 30 minutes, the samples are fully and uniformly mixed to prepare suspension, namely 10^-1And (4) bacterial suspension. Mixing the suspension with sterile water according to a volume ratio of 1: 9 to 10^-2，10^-3，10^-4， 10^-5And (3) taking 0.1mL of bacterial suspension with different dilution times, coating the bacterial suspension on an ISP2 culture medium plate, lightly coating the bacterial suspension on the surface of the culture medium by using a sterile coating rod, standing the bacterial suspension for 30 minutes at room temperature, and then placing the bacterial suspension in a constant-temperature incubator at 28 ℃. And after the bacterial colony grows out, observing and recording the color, the transparency, the surface and the edge form of the bacterial colony. Finally, 1000 strains are picked and inoculated in an ISP2 culture medium to prepare a slant, and fermentation verification is carried out. A strain ring cultured by a slant is picked by using an inoculating loop, and is respectively inoculated into 250mL conical flasks containing 20mL of seed culture medium, after the strain ring is shake-cultured for 1 day at the temperature of 28 ℃, 1mL of strain is further sucked and is transferred into 250mL conical flasks containing 20mL of fermentation medium, after the strain ring is shake-cultured for 3 days at the temperature of 28 ℃, the content of pseudouridine in the obtained fermentation liquid is detected by HPLC, and the most highly productive strain, namely Streptomyces sp HDCC00030 is selected.

Seed culture medium formula (g/L): 10g/L of glucose, 10g/L of corn starch, 10g/L of cottonseed cake powder, 5g/L of yeast extract powder, 15g/L of calcium carbonate, 1.5g/L of magnesium sulfate and 1g/L of monopotassium phosphate, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0 +/-0.1.

Fermentation medium formula (g/L): 10g/L of corn starch, 10g/L of maltodextrin, 20g/L of glucose, 5g/L of yeast extract powder, 10g/L of yeast extract and 30g/L of calcium carbonate, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0 +/-0.1.

Example 2: streptomyces sp HDCC00030 has morphological, cultural, physiological and biochemical characteristics.

Experiments are carried out according to relevant contents in books such as Streptomyces appraisal handbook, Classification and appraisal of actinomycetes, common bacteria system appraisal handbook and the like: the color was determined by reference to the color in the RAL K7 color chart.

1. Morphological characteristics of the strains: the strain HDCC00030 was inoculated into ISP2 medium for plug culture, and after 3-5 days at 28 ℃, cover glass was put on the slide and observed at 400 Xmagnification under an optical microscope, and the results are shown in FIG. 1.

2. The strain culture characteristics are as follows: after the bacterial strain HDCC00030 is cultured on an ISP2 culture medium for 7-10 days at 28 ℃, the colony is elliptical in shape, has radial stripes and a convex surface, the diameter of the colony is about 8-20 mm, the surface of the colony has grooves, the middle of the colony is slightly concave, matrix hypha is developed, the matrix hypha is tightly combined with the culture medium, is not easy to pick up, is beige in color, aerial hypha is white, sporulation is rich, the early stage is white, the later stage is changed into light yellow, no soluble pigment exists, and the result is shown in figure 2.

And for other cultural characteristics, 7 culture media of ISP1, ISP3, ISP4, ISP5, calcium malate, Gao's I and nutrient agar are adopted, and after the culture media are cultured for 7-10 days at the temperature of 28 ℃, the generation conditions of colonies, hyphae, spores and pigments are observed, and the results are shown in Table 2.

TABLE 2 culture characteristics of Strain HDCC00030 on 7 media

3. Physiological and biochemical characteristic test: the results are shown in tables 3 to 8.

a) Utilization of carbon source: using ISP9 as the basal medium, the final concentration of each carbon source was 1.0%, as shown in Table 3.

b) Utilization of inorganic nitrogen source: using ISP9 as the basal medium, the potassium nitrate and ammonium sulfate concentrations were 0.1% each, as shown in Table 3.

c) The basic culture medium adopted in the degradation test and the NaCl tolerance test is GYEA (pH6.8), and the concentration of various degradation products and the degradation test results are shown in Table 4; the results of the NaCl tolerance experiments are shown in Table 8.

d) The catalase test, the pH test and the temperature test all use the ISP2 culture medium. The results of the catalase test are shown in Table 5, the pH test in Table 6, and the temperature test in Table 7.

e) M.R, V-P, etc., the results are shown in Table 5, using the method of "handbook of identifying common bacteria systems".

f) Except for the temperature experiment, the culture is carried out for 7-10 days at 28 ℃.

TABLE 3 utilization of the carbon and nitrogen sources of Strain HDCC00030

TABLE 4 degradation test results of Strain HDCC00030

TABLE 5 major physio-biochemical characteristics of Strain HDCC00030

TABLE 6 pH test for growth of Strain HDCC00030

TABLE 7 temperature test for growth of Strain HDCC00030

Temperature (. degree.C.)	7	14	28	37	45
						Growth conditions	1	1	4	3	0

TABLE 8 tolerance of strain HDCC00030 to NaCl

Remarking: in tables 3 to 8, 0: no growth occurs; 1: the growth is very weak; 2: can grow, has a small amount of spores; 3: the growth is good, and a large number of spores exist; 4: the growth is best, and spores are abundant; +: positive; -: and (4) negativity.

Example 3 species identification

1. 16S rDNA sequence analysis of Streptomyces HDCC00030

Experiments were performed with reference to the book "molecular cloning, laboratory Manual". Mycelia were collected, and then total DNA was extracted using an actinomycete DNA extraction kit. The 16S rDNA sequence amplification is carried out by adopting universal primers 27F (27F: 5'-AGAGTTTGATCCTGGCTCAG-3')/1495R (1495R5 '-CTACGGCTACCTTGTTACGA-3'), the detection of PCR products adopts 0.8 percent agarose gel electrophoresis, the purification and recovery of the PCR products adopt a SanPrep column type PCR purification product kit, and the purified PCR products are directly sent to Nanjing Kingsry Biotech Co.

The 16S rDNA sequence tested by the strain HDCC00030 is compared with the homologous sequence BLAST of related species and genera in a GenBank database after being corrected so as to determine the classification status of the strain.

The 16S rDNA sequence (SEQ ID NO:1) determined by strain HDCC00030 was subjected to BLAST comparison with the relevant sequences in GenBank at the time of NCBI and the results are shown in Table 9 (only the more homologous model strains are shown in the Table).

TABLE 9 homology of Strain HDCC00030 and typical model strains

The 16S rDNA region of the strain HDCC00030(CGMCC NO.21122) is sequenced and compared with the homologous sequence BLAST of related species and genera in a GenBank database, the homology of the strain and Streptomyces (Streptomyces sp. strain TYQ1024, Streptomyces mobaraensis strain DB13, Streptomyces mobaraensis NBRC 13476 and Streptomyces sp. strain KIB-H1992) is up to 99.34-99.64 percent, and the strain HDCC00030 is subjected to an apparent characteristic test, so that the strain HDCC00030 is identified as a Streptomyces sp.

2. The comparison of the Streptomyces HDCC00030 of the invention with other pseudouridine-producing bacteria is as follows:

uematsu T et al reported that Streptomyces ladakanus (Uematsu T, Suhadonik R J. Pseudouridine. isolation and biosynthesis of the nucleotide isolated from the culture broth of Streptomyces ladakanus.12[ J ]. Biochemistry,1972,11(25):4669-4674.) had no description in the literature of the colony morphology and spore color exhibited in the medium, but the concentration of Pseudomonas uridine fermented by Streptomyces ladakanus reported in this document was 70mg/L, which was more different than that of Streptomyces HDCC00030 of the present invention, and when subjected to 16S rDNA comparison, the similarity between Streptomyces hdka 30 and Streptomyces hdka 00030 was not found, and thus the difference was larger.

By combining the morphological, culture, physiological and biochemical characteristics and 16S rDNA sequence identification results of the Streptomyces HDCC00030, the strain HDCC00030 belongs to Streptomyces sp strains, and is different from other known pseudouridine-producing bacteria, so that the Streptomyces HDCC00030 is a brand-new pseudouridine-producing bacteria strain.

Example 4 preparation of pseudouridine fermentation broth

(1) Preparing and culturing slant spores:

slant culture medium formula (g/L): 10g/L of yeast extract powder, 5g/L of malt extract, 10g/L of glucose, 20.0g/L of agar, 7.2-7.4 of pH before digestion, 30 x 200mm of test tube and 15mL of package, sterilizing at 121 ℃ for 20min, cooling to 55-60 ℃, swinging an inclined plane, after cooling and solidification, inoculating a ring of spores or mycelia to the inclined plane, and culturing at 28 +/-1 ℃ for 7-10 days to ensure that the spores are mature.

(2) Preparing and culturing a seed solution:

seed culture medium formula (g/L): 20g/L of glucose, 10g/L of corn starch, 10g/L of soybean cake powder, 5g/L of yeast extract powder, 5g/L of calcium carbonate, 5g/L of magnesium sulfate and 3g/L of dipotassium phosphate. pH7.0 before elimination; a250 mL triangular shake flask with the specification is filled with 50mL and sterilized at 121 ℃ for 20 min. Inoculation 10⁷～10⁸cfu/mL into seed culture medium, 28 + -1 deg.C, 250rpm shaking culture for 48 hr, wherein the culture solution pH is 6.8-7.2, and the mycelium concentration is 15-25% (volume percentage).

(3) Preparing and culturing a fermentation medium:

fermentation medium formula (g/L):

5g/L of corn starch, 5g/L of maltodextrin, 5g/L of glucose, 5g/L of sorbitol, 1g/L of yeast extract powder, 5g/L of soybean cake powder, 5g/L of corn steep liquor dry powder, 1g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate, 1g/L of potassium chloride and 3g/L of calcium carbonate. pH5.0 before digestion. A250 mL triangular shake flask with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 10% (by volume). The culture was carried out at 23. + -. 1 ℃ for 72 hours with shaking at 250 rpm.

The content of pseudouridine in the fermentation broth was determined by HPLC method and found to be 840 mg/L.

Example 5 preparation of pseudouridine fermentation broth

(1) The formulation of the slant culture medium and the culture conditions were the same as those in step (1) of example 4;

(2) the formulation of the seed culture medium and the culture conditions were the same as those in step (2) of example 4;

(3) preparing and culturing a fermentation medium:

20g/L of corn starch, 20g/L of maltodextrin, 30g/L of glucose, 30g/L of sorbitol, 8g/L of yeast extract powder, 10g/L of soybean cake powder, 10g/L of corn steep liquor dry powder, 5g/L of magnesium sulfate, 5g/L of dipotassium hydrogen phosphate, 3g/L of potassium chloride and 20g/L of calcium carbonate. pH6.0 before digestion. A250 mL triangular shake flask with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 10% (by volume). The cells were cultured at 25. + -. 1 ℃ for 120 hours with shaking at 250 rpm.

The content of pseudouridine in the fermentation broth was measured by HPLC method and found to be 1050 mg/L.

Example 6 preparation of pseudouridine fermentation broth

(1) The formulation of the slant culture medium and the culture conditions were the same as those in step (1) of example 4; the formulation and culture conditions of the primary seed medium were the same as those in step (2) of example 4.

(2) Preparing seed liquid in a seeding tank:

the formula of the seed liquid culture medium in the seeding tank is the same as the seed culture medium in the step (2) in the example 4;

putting 10L of seed culture medium into a 15L seed tank, sterilizing by steam at 121 ℃ for 20min, cooling to 28 ℃, and inoculating 200mL of first-stage shake flask seed liquid. Stirring at 200rpm, ventilation of 1.0vvm, and culturing at 28 + -1 deg.C for 48 hr, wherein the pH of the seed solution is 7.0-7.4, and the mycelium concentration is 20-30% (volume ratio).

(3) Preparing fermentation broth of a fermentation tank:

formula of fermentation medium

50g/L of corn starch, 50g/L of maltodextrin, 60g/L of glucose, 50g/L of sorbitol, 15g/L of yeast extract powder, 20g/L of soybean cake powder, 20g/L of corn steep liquor dry powder, 10g/L of magnesium sulfate, 8g/L of dipotassium hydrogen phosphate, 5g/L of potassium chloride and 50g/L of calcium carbonate. pH7.0 before digestion.

The volume of the fermentation tank is 50L, the feeding volume is 30L, the fermentation tank is sterilized by steam, the temperature is 121 ℃, the fermentation tank is 20min, and 3L seed solution in the seed tank is inoculated after the fermentation tank is cooled to 28 ℃. The stirring speed was 300-600rpm (the speed was gradually increased from 300rpm to 600rpm in the first 3 days), the aeration rate was 2.0vvm, and the culture was carried out at 28. + -. 1 ℃ for 168 hours.

The content of pseudouridine in the fermentation broth was found to be 1360mg/L by HPLC method.

Sequence listing

<110> Zhejiang a kind of jade Biotech Co., Ltd

<120> streptomycete and method for producing pseudouridine by fermentation of streptomycete

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1419

<212> DNA

<213> Streptomyces sp.

<400> 1

caccgagcgc gtgtacctgc agtcgacgat gacctctttc gggaggggat tagtggcgaa 60

cgggtgagta acacgtgggc aatctgccct gcactctggg acaagccctg gaaacggggt 120

ctaataccgg atacgactac tgaccgcatg gttggtggtg gaaagctccg gcggtgcagg 180

atgagcccgc ggcctatcag cttgttggtg gggtgatggc ctaccaaggc gacgacgggt 240

agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 300

gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 360

gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgag agtgacggta 420

cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcaa 480

gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gcttgtcgcg tcggatgtga 540

aagcccgggg cttaaccccg ggtctgcatt cgatacgggc aggctagagt tcggtagggg 600

agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 660

aaggcggatc tctgggccga tactgacgct gaggagcgaa agcgtgggga gcgaacagga 720

ttagataccc tggtagtcca cgccgtaaac gttgggaact aggtgtgggc gacattccac 780

gtcgtccgtg ccgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct 840

aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggc ttaattcgac 900

gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aagcgctaga gatagtgccc 960

cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1020

ggttaagtcc cgcaacgagc gcaacccttg ttctgtgttg ccagcatgcc tttcggggtg 1080

atggggactc acaggagact gccggggtca actcggagga aggtggggac gacgtcaagt 1140

catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggccggtac aatgagctgc 1200

gataccgtga ggtggagcga atctcaaaaa gccggtctca gttcggattg gggtctgcaa 1260

ctcgacccca tgaagttgga gttgctagta atcgcagatc agcattgctg cggtgaatac 1320

gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca cccgaagccg 1380

gtggcccaac ccttgtggag ggagcgtcga agtacgtcc 1419

Claims

1. Streptomyces sp HDCC00030 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.21122 and the preservation date of 2020, 11 and 6 days.

2. Use of Streptomyces sp HDCC00030 according to claim 1 for the preparation of pseudouridine or pharmaceutical compositions containing pseudouridine.

3. A method for preparing pseudouridine, which is characterized by comprising the following steps: comprising the step of aerobic fermentation using a Streptomyces sp HDCC00030 according to claim 1 in a nutrient medium containing assimilable carbon and/or nitrogen sources.

4. The method of claim 3, wherein: the assimilable carbon source is selected from one of corn starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or the combination of the substances; preferably one of corn starch, maltodextrin, glucose, sucrose and sorbitol or a combination of any of the above substances.

5. The method of claim 3, wherein: the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea and ammonium salt or the combination of the above substances; preferably soybean cake powder, yeast extract powder, corn steep liquor dry powder, yeast powder or a combination of any of the above substances.

6. The method of claim 3, wherein: the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from one of trisodium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate or the combination of any of the substances; preferably one or the combination of any several of calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate and potassium chloride.

7. The method of claim 3, wherein: the nutrient medium contains 5-50g/L of corn starch, 5-50g/L of maltodextrin, 5-60g/L of glucose, 5-50g/L of sorbitol, 1-15g/L of yeast extract powder, 5-20g/L of soybean cake powder, 5-20g/L of corn steep liquor dry powder, 1-10g/L of magnesium sulfate, 2-8g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium chloride and 3-50g/L of calcium carbonate.

8. The method of claim 3, wherein: the temperature of the aerobic fermentation is 20-35 ℃, preferably 20-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24-240 hours, preferably 72-168 hours; the oxygen throughput is from 0.1 to 2.0vvm, preferably from 0.5 to 2.0 vvm.

9. The method according to any one of claims 3-8, wherein: the Streptomyces sp HDCC00030 is inoculated into the nutrient medium through seed liquid to carry out fermentation culture; the seed solution is obtained by seed culture of the Streptomyces sp HDCC00030 in a seed culture medium.

10. The method of claim 9, wherein: the seed culture medium contains 5-30g/L of glucose, 5-20g/L of corn starch, 2-10g/L of soybean cake powder, 1-10g/L of yeast extract powder, 1-20g/L of calcium carbonate, 1-10g/L of magnesium sulfate and 1-10g/L of dipotassium phosphate.

11. The method of claim 10, wherein: the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, and the optimal temperature is 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24 to 80 hours, preferably 24 to 60 hours.