CN113174344B

CN113174344B - Strain for producing tunicacidin B and application thereof

Info

Publication number: CN113174344B
Application number: CN202110461991.3A
Authority: CN
Inventors: 朱进伟; 郑玲辉; 匡春兰; 彭湘屏; 张敏; 石磊; 孙琼; 高祥; 陈伟; 汪超
Original assignee: Zhejiang Hunda Biotechnology Co ltd
Current assignee: Zhejiang Hunda Biotechnology Co ltd
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2023-01-31
Anticipated expiration: 2041-04-27
Also published as: CN113174344A

Abstract

The invention discloses a Tistrella mobilis (HDCC 00053) which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.22003. The Stachytrid motilis HDCC00053 (CGMCC NO. 22003) is a brand-new strain of the phaeophytin B producing strain, the production capacity is high, the capacity of producing the phaeophytin B is greatly improved compared with other strains in the prior art, the titer of the phaeophytin B can reach more than 300mg/L, and the industrial production is favorably realized.

Description

Strain for producing tunicacidin B and application thereof

Technical Field

The invention relates to the technical field of industrial microbial fermentation, in particular to a strain for producing a tunicaicin B and application thereof.

Background

Tunicasin B (didemnin B) was found in Didemnum solidum, an evolutionarily low protozoon living from the Caribbean territory in 1981 by professor Rinehart, university of Illinois USA. Moesin B belongs to cyclic depsipeptides and was introduced in phase I clinical trials in 1984 (developed by fevered, once in phase ii for the treatment of brain tumors, malignancies and non-hodgkin lymphoma), the 1 st marine natural product that was introduced in clinical studies in the united states. In vivo screening results show that the membrane ecteinascidin B has strong activity for resisting P388 leukemia and B16 melanoma, can induce rapid apoptosis of HL-60 tumor cells and apoptosis of a plurality of transformed cells, and further pharmacological research shows that the IC50 of the compound on L1210 leukemia cells is 2ng/mL. At present, a series of researches are carried out on the compound and more derivatives, and antitumor drugs with better curative effect and lower toxic and side effects are expected to be found.

Due to the biological and chemical properties of tunicacidin B, in the development of related anti-tumor bioactivity test experiments and obtaining more derivatives, a large amount of corresponding tunicacidin B samples need to be prepared to satisfy various preclinical activity studies, and at present, the reported preparation methods of tunicacidin B mainly comprise a chemical Synthesis method and a microbial fermentation method, wherein the literatures effective Total Synthesis of Didermins A and B and Total Synthesis of Didermins A, B and C report a chemical Synthesis method, but the Synthesis method only stays in a laboratory stage, and the chemical Synthesis has the problems of long reaction steps and low yield. The prior art reports that the potency level of the microbial fermentation for producing the membrane sea squirol B is generally low, and JP2012240974A reports a strain of motile Citrella mobilis YIT12409 (FERM AP-22080), the strain has the capability of producing the membrane sea squirol B, and the potency is calculated to be about 20mg/L. The potency of α -proteobacterium Tistrella molis membrane-producing ecteinascidin B reported in the Bacterial Production of the tubular inhibitor Cyclic Depsipeptide Didemnin B is about 3.2mg/L; the literature Bacterial biosyntheses and administration of the Didemnin Anti-canceragens also reports low yields of 0.2mg/L by fermentation of the microorganism T.mobilis KA 081020-065.

In summary, aiming at the problems of low fermentation yield, long synthesis steps, low yield, and low safety in the chemical synthesis preparation process of the mojaponin B obtained by fermentation in the prior art, the invention seeks a new microorganism, and can obtain the mojaponin B with high fermentation yield by simple fermentation, thereby realizing low production cost of the mojaponin B.

Disclosure of Invention

In order to solve the problem of insufficient conventional preparation method of the tunicaridin B, one of the purposes of the present invention is to provide a novel tunicaridin B-producing strain, which is Tistrella mobilis (HDCC 00053), which is preserved in China general microbiological culture Collection center (address: no. 3 of West Lu 1 of North Chen Such. No. 3 of facing-Yang district, china institute of microbiology, china academy of sciences) in 2021, 12 days, with the preservation number of CGMCC NO.22003, and registered in a book to prove survival.

The nucleotide sequence of the strain is shown as SEQ ID NO. 1.

The invention also aims to provide application of Tistrella mobilis (HDCC 00053 (CGMCC NO. 22003)) in preparation of the hymenacin B.

The invention also provides a preparation method of the hymenacidin B, which comprises the step of carrying out aerobic fermentation on the hymenacidin B in a nutrient medium containing a carbon source, a nitrogen source and inorganic salts by adopting Tistrella mobilis (HDCC 00053 (CGMCC NO. 22003).

In the embodiment of the present invention, the carbon source is selected from glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, sorbitol, or a combination of any of them, more preferably glycerol, dextrin, starch, lactose, or a combination of any of them, and even more preferably glycerol, dextrin, or a combination of them.

In a preferred embodiment, the nitrogen source is selected from yeast nitrogen source (including but not limited to yeast powder, yeast extract, yeast peptone), peptone (including vegetable peptone or animal peptone), casein, soy protein isolate, soy protein concentrate, pea protein, tyrosine, soybean (cake) powder, cottonseed cake powder, corn gluten powder, peanut cake powder, urea, (nitrite) nitrate, ammonium salts or a combination of any of them; preferably yeast nitrogen source (including but not limited to yeast powder, yeast extract, yeast peptone), peptone (including vegetable peptone and animal peptone), casein, soy protein isolate, soy protein concentrate, pea protein, tyrosine or any combination thereof; more preferably yeast nitrogen source (including but not limited to yeast powder, yeast extract, yeast peptone), peptone (including vegetable peptone or animal peptone), tyrosine or any combination thereof.

In a preferred embodiment, the inorganic salt is selected from the group consisting of seawater, sodium chloride, acetate, sulfate, nitrate, and magnesium, cobalt, zinc, (ferrous) manganese, copper, molybdate ions, or a combination of any of several; preferably seawater crystal, sodium chloride, acetate, sulfate, magnesium ions, cobalt ions, zinc ions or a combination of any of the seawater crystal, the sodium chloride, the acetate and the sulfate; more preferably sea crystal, sodium chloride, sodium acetate, magnesium sulfate, zinc sulfate, cobalt chloride or a combination of any of them.

In a preferred embodiment, the nutrient medium comprises or consists of the following components in weight percent: 2 to 7 percent of lactose, 0 to 2 percent of glucose, 1 to 10 percent of glycerol, 0 to 8 percent of dextrin, 0.5 to 3 percent of yeast extract powder, 0.5 to 4 percent of peptone, 0.05 to 0.4 percent of magnesium sulfate, 0.2 to 3 percent of sodium chloride, 0 to 3 percent of sea crystal, 0.1 to 2 percent of tyrosine, 0 to 0.001 percent of cobalt chloride, 0 to 0.001 percent of zinc sulfate, 0 to 0.1 percent of defoaming agent and the balance of water; more preferably: 4% of lactose, 1% of glucose, 2% of glycerol, 1% of yeast extract powder, 1.5% of peptone, 0.2% of magnesium sulfate, 2% of sodium chloride, 0.0005% of cobalt chloride, 0.0004% of zinc sulfate, 0.05% of defoaming agent and the balance of water; the glucose, dextrin, sea crystal, cobalt chloride, zinc sulfate and defoamer can be 0.

In a preferred embodiment, the fermentation temperature is 25 to 32 ℃, and more preferably 28 to 30 ℃; 5 to 40 percent of dissolved oxygen, and more preferably 5 to 15 percent; the pH is controlled to be 6.8 to 8.8, and the preferable pH is 7.0 to 7.8; the fermentation culture time is 3 to 14 days, and more preferably 3 to 7 days.

In a preferred embodiment, starting on day 2 of the fermentation process, feeding carbon and nitrogen sources to the fermentation medium is added when the glycerol concentration is below 1.0% or the dissolved ammonium is below 400 ppm; it is further preferred that the daily average feed concentration of the carbon source feed (including but not limited to glycerol, lactose or a combination thereof) is 1 to 3% and the daily average feed concentration of the nitrogen source feed (including but not limited to yeast extract powder, peptone or a combination thereof) is 0.3 to 1.5%.

In a preferred embodiment, the fermentation culture is carried out by inoculating the Statrimaran mobilis (Tistrella mobilis) HDCC00053 (CGMCC NO. 22003) to a fermentation medium after seed culture.

In a preferred embodiment, the inoculum size of the fermentation is 2 to 20%, more preferably 4 to 10%.

In a preferred embodiment, the seed medium comprises or consists of a carbon source, a nitrogen source and inorganic salts.

In a preferred embodiment, the carbon source is selected from galactose, glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, sorbitol, or a combination of any of them, further preferably glycerol, dextrin, lactose, or a combination of any of them, and more preferably glycerol, lactose, or a combination of them.

In a preferred embodiment, the nitrogen source is selected from a yeast nitrogen source (including yeast powder, yeast extract, yeast peptone), peptone (including vegetable peptone and animal peptone), casein, soy isolate protein, soy concentrate protein, pea protein or a combination of any of the above, preferably a yeast nitrogen source (including yeast powder, yeast extract powder, yeast peptone), peptone (including vegetable peptone and animal peptone) or a combination of any of the above, and more preferably yeast extract powder, peptone or a combination of the above.

In a preferred embodiment, the inorganic salt is selected from sodium chloride (or its equivalent alternatives including, but not limited to, sea salt, sea rock), sodium acetate, or combinations thereof.

In a preferred embodiment, the seed culture medium comprises or consists of: 0 to 3 percent of glycerol, 0 to 1.5 percent of glucose, 0 to 1.5 percent of lactose, 0.2 to 2 percent of yeast organic nitrogen source, 0 to 2 percent of peptone, 0.1 to 2 percent of sodium chloride and 0 to 1 percent of sodium acetate; more preferably: 1.2 to 1.8 percent of lactose, 0.4 to 0.8 percent of glucose, 0.6 percent of yeast extract powder, 1 percent of peptone, 2 percent of sodium chloride, 0 to 0.1 percent of defoaming agent and the balance of water.

In a preferred embodiment, the seed medium comprises a primary seed medium and a secondary seed medium, which may be the same or different; further, the first-order seed culture medium is preferably 1.2% of lactose, 0.4% of glucose, 0.6% of yeast extract powder, 1% of peptone, 2% of sodium chloride and the balance of water; further, the secondary seed culture medium comprises 1.8% of lactose, 0.8% of glucose, 0.5% of yeast extract powder, 1.5% of peptone, 2% of sodium chloride, 0.05% of an antifoaming agent and the balance of water.

In a preferred embodiment, during said primary seed culture: the inoculation amount is more than or equal to 1 percent, and the preferable inoculation amount is 4 percent; the temperature is 25 to 34 ℃, and the more preferable temperature is 28 to 30 ℃; the loading capacity is 50 to 1000ml, and more preferably 400 to 700ml; the rotating speed is more than or equal to 200rpm, and more preferably 220-270 rpm; the culture period is 20 to 54 hours, and more preferably 40 to 48 hours.

In a preferred embodiment, during said secondary seed culture: the inoculation amount is more than or equal to 0.03 percent, and the more preferable range is 0.1 to 0.5 percent; the temperature is 25 to 34 ℃, and the more preferable temperature is 28 to 30 ℃; dissolved oxygen is more than or equal to 25 percent, and more preferably more than or equal to 35 percent; the pH value is more than or equal to 6.5, and more preferably 6.8-7.8; the culture period is 20 to 54 hours, and more preferably 20 to 48 hours.

Further, more specifically, the seed movement tiustralis HDCC00053 (CGMCC NO. 22003) is inoculated to a slant culture for activation (25-34 ℃ for 2-5 d) before seed culture, and bacterial lawn is washed down by sterile water and scattered to prepare bacterial suspension.

The slant (solid) culture medium can be lactose, glycerol, glucose, yeast organic nitrogen source, peptone, sodium chloride (or its equivalent substitute), agar combination, such as lactose 0.4%, glycerol 0.4%, yeast extract powder 0.6%, peptone 1%, sodium chloride 2%, agar 1.8%. pH7.0, sterilizing at 121 deg.C for 30min.

The strain of Tistrella mobilis (HDCC 00053) has the characteristics that: the colony is about 3-5mm, white or grey white, opaque, smooth and moist surface or few wrinkles, round and regular edge, gram negative.

The invention protects the hymenicidin B production strain HDCC00053 (CGMCC NO. 22003) which is a brand-new hymenicidin B producing strain, has high production capacity, greatly improves the capacity of producing the hymenicidin B compared with other strains in the prior art, ensures that the titer of the hymenicidin B can reach more than 300mg/L, and is beneficial to realizing industrial production.

Drawings

FIG. 1 is HPLC chromatogram of capacity identification fermentation liquor of CGMCC NO.22003 strain;

FIG. 2 is LCMS spectrum of capability identification fermentation liquor of CGMCC NO.22003 strain;

FIG. 3 is HPLC chromatogram of shake flask fermentation liquid of CGMCC NO.22003 strain optimization process;

FIG. 4 is HPLC chromatogram of fermentation liquor of CGMCC NO.22003 strain optimization process tank.

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.

The present invention is further described below in conjunction with specific examples, which are intended to illustrate the invention and not to limit the scope of the invention.

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

In the following examples, the LCMS and HPLC methods were used to detect tunicamycin B in the fermentation broth, both using the starchacin B standard as a control, and the detection conditions of the LCMS were as follows:

equipment: LC-1260-MS-6540QTOF

And (3) chromatographic column: c18 150X 4.6mm,1.8 μm

Wavelength: 210nm

Column temperature: 40 deg.C

Flow rate: 1ml/min

Sample introduction amount: 5 μ l

Operation: for 10min

Mobile phase: 59% acetonitrile

The HPLC detection conditions were as follows:

a chromatographic column: c18 50X 4.6mm,1.8 μm

Wavelength: 210nm

Column temperature: 40 deg.C

Flow rate: 1ml/min

Sample injection amount: 5 μ l

Operation: 5min

Mobile phase: 59% acetonitrile

EXAMPLE 1 Strain origin

From the sea area near the east polar island of Zhoushan, zhejiang, 34 samples of surface seawater, deep seawater (about 50 m), bottom seawater and seabed sediment (light yellow) were collected by a multi-tube sampler. Inoculating 5ml (g) of each sample to an enrichment medium, performing light-shielding oscillation culture at 28 ℃ and 220rpm for 5d, performing gradient dilution by using sterile normal saline, coating the diluted sample on a 2216E marine agar medium, performing light-shielding culture at 28 ℃ and 70% humidity for 3d, selecting gray-white, smooth, wrinkle-free and regular round colonies on the surface, respectively preserving the colonies, simultaneously performing amplification culture at 28 ℃ and 220rpm by using liquid 2216E marine culture, inoculating the colonies to a shaking flask primary screening medium (1% galactose, 1.5% peptone, 1% glucose and 0.2% yeast extract powder), performing culture at 28 ℃ and 220rpm, performing detection by using HPLC after pretreatment of samples in different culture periods, and comparing the samples by using a reference substance. The results show that components with the same relative retention time as the reference substance are detected in the fermentation liquor cultured for 4-7 days by the TM264 strain and the TM887 strain, and the molecular weight is determined by LCMS and matched with the reference substance, and the components are 1112. From fermentation titer evaluation, the production capacity of the strain TM887 is obviously higher than that of the strain TM264, so the strain TM887 is selected as a production strain, an original number HDCC00053 is given, the strain is inoculated on an inclined plane and placed in a constant temperature box with 28 ℃ and 55% humidity for culture for 3 days, and then the strain is collected for 16S rRNA sequencing, and the result shows that the strain has 99% homology with the 16S rRNA sequence of Tistrella mobilis KA081020-065 and is determined to be the Tistrella mobilis. The original number HDCC00053 strain slant is preserved in China general microbiological culture Collection center (CGMCC) at 12.03.2021 with the preservation number of CGMCC NO.22003. The preservation slant culture medium comprises the following components: 0.4% of lactose, 0.4% of glycerol, 0.6% of yeast extract powder, 1% of peptone, 2% of sodium chloride and 1.8% of agar.

Example 2CGMCC NO.22003 Strain physiological and biochemical tests

The physiological and biochemical experiments are carried out according to the methods in reference books such as Bergey's Manual of identification of bacteria and Manual of identification of common bacteria systems. In this example, the culture was carried out at 28 ℃ for 3 to 10 days in all the tests except the temperature test.

Morphological characteristics:

the CGMCC NO.22003 strain glycerol tube suspension is diluted to 10 in a gradient way ^-5 And separating, coating and inoculating to an improved plate culture medium, culturing in a constant temperature box with 28 ℃ and 55% humidity for 5 days, and observing the morphological characteristics of colonies. Finally, mature colonies were found to be about 3-5mm in size, white or off-white, opaque, smooth and moist on the surface or with few wrinkles, round and regular edges, gram-negative.

The formula of the plate culture medium comprises: 0.4% of lactose, 0.4% of glycerol, 0.6% of yeast extract powder, 1% of peptone, 2% of sodium chloride and 1.8% of agar. pH7.0, sterilizing at 121 deg.C for 30min.

Physiological and biochemical characteristics: see tables 1-5 for details.

TABLE 1 utilization of carbon and nitrogen sources of CGMCC NO.22003 Strain

TABLE 2 main physiological and biochemical characteristics of CGMCC NO.22003 strain

Test items	Results	Test items	As a result, the
				Liquefaction of gelatin	+	Milk peptone	-
Starch hydrolysis	+	Nitrate reduction	+
				Arginine hydrolysis	+	Indoles	+
Oxidase enzyme	+	v.P experiment	+
				Catalase enzyme	+	M.R. experiment	-
Beta-galactosidase enzyme	+	/	/

TABLE 3 CGMCC NO.22003 Strain growth pH test

pH	2.0	3.0	4.0	5.0	6.0	7.0	8.0	9.0
									Growth conditions	0	0	1	1	2	4	4	3

TABLE 4 CGMCC NO.22003 Strain growth temperature test

Temperature (. Degree.C.)	7	14	25	28	37	45
							Growth conditions	0	0	3	4	4	0

TABLE 5 tolerance of CGMCC NO.22003 to NaCl

NaCl concentration	1％	4％	7％	10％
					Growth of the strain	3	2	0	0

* Note: 0, no growth; 1, very weak growth; 2, growth can be realized; 3, good growth; 4, the best growth is achieved; positive; -, negative; w, weak.

Example 3 confirmation of the ability of CGMCC NO.22003 Strain to produce moesin B

1) Taking separated and purified CGMCC NO.22003 strain working strain glycerin tube, thawing, and diluting with sterile normal saline to 10 ^-2 0.1ml of diluent is absorbed and inoculated to the 2216E marine agar culture medium inclined plane and evenly coated, and the inclined plane lawn is obtained after the cultivation for 3 days in the dark under the conditions of 28 ℃ and 70 percent of relative humidity.

2) Taking a mature culture inclined plane, adding 10ml of sterile normal saline, scraping off the lawn, and scattering to obtain a bacterial suspension.

3) 8ml of the bacterial suspension is sucked up and inoculated into a 2800ml triangular flask containing 400ml of shake flask fermentation medium, and after being bundled, the mixture is placed on a shaking table at 25 ℃ and 140rpm for shaking culture for 14 days.

Wherein, the formula of the shake flask fermentation culture medium comprises: 0.1% galactose, 0.1% gelatin peptone, 0.1% glycerol, 0.15% yeast extract powder, 0.5% peptone, 3% XAD-7 resin.

4) After the shake flask fermentation is finished, centrifuging 400ml of shake flask fermentation liquor, collecting thalli and XAD-7 resin, oscillating and soaking for 2h by using 200ml of anhydrous methanol, centrifuging, collecting supernate, continuously oscillating and soaking the mushroom dregs and the resin for 2h by using 200ml of fresh anhydrous methanol, centrifuging, and collecting supernate. And mixing the two supernatants, concentrating by rotary evaporation to obtain a paste, adding 200ml of ethyl acetate for redissolving, separating to obtain an ethyl acetate phase, concentrating by rotary evaporation again to dryness, and redissolving by 40ml of anhydrous methanol. 2ml of the re-solution was filtered through a 0.45 μm filter membrane and analyzed and detected by HPLC and LCMS.

5) HPLC and LCMS detection spectra are shown in figure 1 and figure 2 respectively. HPLC and LCMS analysis confirmed that the fermentation broth contained sphingosine B (with an accurate molecular weight of 1111.64), and the content of sphingosine B was 10.3. Mu.g/ml as converted to a control.

Example 4 preparation of Membrane Colostrinin B by Shake flask fermentation

1) Taking separated and purified CGMCC NO.22003 strain working strain glycerin tube, thawing, and diluting with sterile normal saline to 10 ^-2 Sucking 0.1ml of diluent, inoculating to an improved slant culture medium, uniformly coating, and culturing in dark at 28 ℃ and 70% relative humidity for 3d to obtain slant lawn.

3) 10ml of the bacterial suspension is sucked and inoculated into an optimized shake flask seed culture medium containing 500ml, and after being bundled, the mixture is placed on a shaking table with the temperature of 30 ℃ and the rpm of 220 to be cultured by shaking for 2 days.

Wherein, the optimized shake flask seed culture medium formula comprises: 1.2% of lactose, 0.4% of glucose, 0.6% of yeast extract powder, 1% of peptone and 2% of sodium chloride.

4) After the seeds are cultured to be mature, absorbing 4ml of seed culture solution, inoculating the seed culture solution into an optimized shake flask fermentation culture medium containing 100ml, and placing the seed culture solution on a shaking table with the temperature of 30 ℃ and the rpm of 220 for shaking culture for 7 days after the seed culture solution is bundled.

Wherein, the optimized shake flask fermentation medium formula comprises: lactose 5%, glucose 2%, starch 1.5%, yeast extract powder 1%, peptone 1%, sodium acetate 0.5%, magnesium sulfate 0.2%, sodium chloride 2%, cobalt chloride 0.0005%, and zinc sulfate 0.0004%.

5) After the shake flask fermentation is finished, taking 1mL of fermentation liquor, adding 3mL of anhydrous methanol, carrying out ultrasonic treatment for 2h after uniform mixing, centrifuging for 10min at 14000rpm after uniform mixing again, filtering by using a 0.45 mu m filter membrane, and carrying out quantitative analysis by using HPLC (high performance liquid chromatography), wherein the content of the film-shaped ecteinascidin B in the fermentation liquor is 307 mu g/mL, and the HPLC chromatogram of the fermentation liquor is shown in figure 4.

EXAMPLE 5 preparation of Coleophytin B by tank fermentation

3) 10ml of the bacterial suspension is sucked and inoculated into an optimized primary shake flask seed culture medium containing 500ml, and after being bundled, the mixture is placed on a shaking table with the temperature of 30 ℃ and the rpm of 220 to be shake-cultured for 2 days.

Wherein, the optimized first-level shake flask seed culture medium formula comprises: 1.2% of lactose, 0.4% of glucose, 0.6% of yeast extract powder, 1% of peptone and 2% of sodium chloride.

4) After the first-level seeds are cultured and matured, 50ml of seed culture solution is taken and inoculated into 10L of optimized second-level seed culture medium (a seed tank), the culture temperature is 28-30 ℃, the dissolved oxygen is controlled to be not less than 35 percent, the pH is controlled to be 6.8-7.8, and the culture period is 42h.

Wherein, the optimized secondary seed culture medium formula comprises: 1.8% of lactose, 0.8% of glucose, 0.5% of yeast extract powder, 1.5% of peptone, 2% of sodium chloride and 0.05% of defoaming agent.

5) After the secondary seeds are cultured and matured, inoculating the secondary seed liquid into an optimized tank fermentation medium according to the proportion of 10%, wherein the fermentation temperature is 28-30 ℃, the initial stirring speed is 150rpm, the air flow is 0.5vvm, the dissolved oxygen linkage control range is 5-15%, and the pH acid-base combined control is 7.0-7.8. After 2 days of fermentation culture, feeding is started, the daily average feeding concentration of a carbon source (mixed by glycerol and lactose) is 1-3%, the daily average feeding concentration of peptone is 0.3-1.5%, and the fermentation culture is finished until 7 days.

Wherein, the optimized tank fermentation medium formula comprises: lactose 4%, glucose 1%, glycerol 2%, yeast extract powder 1%, peptone 1.5%, magnesium sulfate 0.2%, sodium chloride 2%, cobalt chloride 0.0005%, zinc sulfate 0.0004%, and defoaming agent 0.05%. The optimized feed medium formula comprises: 30% glycerol, 10% lactose, 15% peptone.

6) After the tank fermentation is finished, taking 1mL of fermentation liquor, adding 3mL of anhydrous methanol, carrying out ultrasonic treatment for 2h after uniform mixing, centrifuging for 10min at 14000rpm after uniform mixing again, filtering by using a 0.45 mu m filter membrane, and carrying out quantitative analysis by using HPLC (high performance liquid chromatography), wherein the content of the tundinin B in the fermentation liquor is 242 mu g/mL, and the HPLC chromatogram of the fermentation liquor is shown in figure 4.

Sequence listing

<110> Zhejiang \29682

<120> a strain for producing tunicacidin B and application thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1360

<212> DNA/RNA

<213> Tistrella mobilis

<400> 1

ctgcctccct tacgggtcag ctcaccggct tcgggtaaac caactcccat ggtgtgacgg 60

gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgttccgc gattactagc 120

gattccgact tcatgcactc gagttgcaga gtacaatccg aactgagacg actttttgag 180

attagcttca cctcgcgatg tcgctgccca ctgtagtcgc cattgtagca cgtgtgtagc 240

ccagcccata agggccatga ggacttgacg tcatccccac cttcctccgg cttatcaccg 300

gcagtttccc tagagtgccc agccgaactg atggcaacta aggatgaggg ttgcgctcgt 360

tgcgggactt aacccaacat ctcacgacac gagctgacga cagccatgca gcacctgtgt 420

gacgtccggc cgaaccgaaa accccgtctc cagggtcgcg acgtccatgt caagggctgg 480

taaggttctg cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540

tcaattcctt tgagttttaa ccttgcggcc gtactcccca ggcggagtgc ttaacgcgtt 600

agctacgaca ctgcgatact aagtatccca acgtccagca ctcatcgttt acggcgtgga 660

ctaccagggt atctaatcct gtttgctccc cacgctttcg tgcctcagcg tcagtttcgg 720

gccaggcagc cgccttcgcc accggtgttc ttcccaatat ctacgaattt cacctctaca 780

ctgggaattc cactaccctc tcccgaactc cagcctacca gtctcaaaag cagttccgga 840

gttgagcccc gggctttcac ttctgacttg ataagccgcc tacgcacgct ttacgcccag 900

taaatccgaa caacgctagc ccccttcgta ttaccgcggc tgctggcacg aagttagccg 960

gggcttcttc tacgggtacc gtcattatct tccccgtcga aagagcttta caatccgaag 1020

accttcatca ctcacgcggc attgctggat caggctttcg cccattgtcc aatattcccc 1080

actgctgcct cccgtaggag tctgggccgt gtctcagtcc cagtgtggct gatcatcctc 1140

tcagaccagc taccgatcgt cgccttggta ggccgttacc ccaccaacta gctaatcgga 1200

cgcgggctca tccatctacg gccgaagcct ttcccccgaa gggcgtatgc ggtattagct 1260

gcagtttccc gcagttattc cccatagatg ggcagattcc cacgtgttac tcacccgtgc 1320

gccacgtaag ccggaaccga agttccgacc ccgtcgactg 1360

Claims

1. A sports Testrina (A. Scriptus)Tistrellamobilis) HDCC00053, preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC NO.22003 and the preservation date of 2021, 3 months and 12 days.

2. The motile Testrina strain(s) of claim 1Tistrellamobilis) HDCC00053 (CGMCC NO. 22003) in preparationApplication of prepared tunicacidin B is provided.

3. A method for preparing a tunicacidin B is characterized by comprising the following steps: the method comprises using the Standula mobilis (Standula mobilis) (of claim 1)Tistrellamobilis) HDCC00053 is obtained by aerobic fermentation in a nutrient medium containing carbon source, nitrogen source and inorganic salts.

4. The method of claim 3, wherein: the carbon source is selected from glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, sorbitol or a combination of any of the above.

5. The method of claim 4, wherein: the carbon source is selected from glycerol, dextrin, starch, lactose or the combination of any of the glycerol, the dextrin, the starch and the lactose.

6. The method of claim 5, wherein: the carbon source is selected from glycerol, dextrin or a combination of the glycerol and the dextrin.

7. The method of claim 3, wherein: the nitrogen source is selected from yeast nitrogen source, peptone, casein, soybean protein isolate, soybean protein concentrate, pea protein, tyrosine, soybean cake, soybean meal, cottonseed cake meal, corn protein meal, peanut cake meal, urea, nitrite, nitrate, ammonium salt or the combination of any of the above.

8. The method of claim 7, wherein: the nitrogen source is selected from yeast nitrogen source, peptone, casein, soy protein isolate, soy protein concentrate, pea protein, tyrosine or any combination thereof.

9. The method of claim 8, wherein: the nitrogen source is selected from yeast nitrogen source, peptone, tyrosine or any combination of the yeast nitrogen source, the peptone and the tyrosine.

10. The method of claim 9, wherein: the nitrogen source is selected from yeast powder, yeast extract and yeast peptone.

11. The method of claim 3, wherein: the nutrient medium also comprises inorganic salt, and the inorganic salt is selected from seawater crystal, sodium chloride, acetate, sulfate, nitrate or magnesium, cobalt, zinc, iron, ferrous iron, manganese, copper, molybdate ions or the combination of any of the above.

12. The method of claim 11, wherein: the inorganic salt is selected from seawater crystal, sodium chloride, acetate, sulfate, magnesium ions, cobalt ions, zinc ions or a combination of any of the above.

13. The method of claim 12, wherein: the inorganic salt is selected from seawater crystal, sodium chloride, sodium acetate, magnesium sulfate, zinc sulfate, cobalt chloride or the combination of any of the above.

14. The method of claim 3, wherein: the nutrient medium comprises or consists of: 2 to 7 percent of lactose, 0 to 2 percent of glucose, 1 to 10 percent of glycerol, 0 to 8 percent of dextrin, 0.5 to 3 percent of yeast extract powder, 0.5 to 4 percent of peptone, 0.05 to 0.4 percent of magnesium sulfate, 0.2 to 3 percent of sodium chloride, 0 to 3 percent of sea crystal, 0.1 to 2 percent of tyrosine, 0 to 0.001 percent of cobalt chloride, 0 to 0.001 percent of zinc sulfate and 0 to 0.1 percent of defoaming agent.

15. The method of claim 14, wherein: the nutrient medium comprises or consists of: lactose 4%, glucose 1%, glycerol 2%, yeast extract powder 1%, peptone 1.5%, magnesium sulfate 0.2%, sodium chloride 2%, cobalt chloride 0.0005%, zinc sulfate 0.0004%, and defoaming agent 0.05%.

16. The method of claim 3, wherein: the fermentation temperature is 25 to 32 ℃; dissolved oxygen is 5 to 40 percent; controlling the pH value to be 6.8 to 8.8; the fermentation culture time is 3 to 14 days.

17. The method of claim 16, wherein: the fermentation temperature is 28 to 30 ℃; dissolved oxygen is 5 to 15 percent; controlling the pH value to be 7.0 to 7.8; the fermentation culture time is 3 to 7 days.

18. The method of claim 3, wherein: also comprises the step of adding a supplemented carbon source and a nitrogen source into the fermentation medium in the fermentation process.

19. The method of claim 18, wherein: the daily average fed-batch concentration of the fed-batch carbon source is 1 to 3 percent, and the fed-batch carbon source is glycerol, lactose or a combination thereof; the daily average feeding concentration of the feeding nitrogen source is 0.3-1.5%, and the feeding nitrogen source is yeast extraction powder, peptone or a combination thereof.

20. The method of claim 3, wherein: the described Testurina mobilis (A)Tistrellamobilis) HDCC00053 (CGMCC NO. 22003) is inoculated to a fermentation medium through a seed solution for fermentation culture; the seed liquid is the sports Testrina (R) of claim 1Tistrellamobilis) HDCC00053 (CGMCC NO. 22003) is obtained by seed culture in a seed culture medium.

21. The method of claim 20, wherein: the seed culture medium comprises or consists of a carbon source, a nitrogen source and inorganic salts; the carbon source is selected from galactose, glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, sorbitol or the combination of any of the galactose, the glycerol, the dextrin, the starch, the glucose, the lactose, the sucrose, the maltose, the mannitol and the sorbitol; the nitrogen source is selected from yeast nitrogen source, peptone, casein, soy protein isolate, soy protein concentrate, pea protein or any combination thereof.

22. The method of claim 20, wherein: the carbon source is selected from glycerol, dextrin, lactose or the combination of any of the glycerol, the dextrin and the lactose; the nitrogen source is selected from yeast nitrogen source, peptone or the combination of any several of the nitrogen sources.

23. The method of claim 22, wherein: the carbon source is selected from glycerol, lactose or a combination of the glycerol and the lactose; the nitrogen source is selected from yeast extract powder, peptone or a combination of the yeast extract powder and the peptone.

24. The method of claim 20, wherein: the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from sodium chloride, sea salt, seawater crystal, sodium acetate or the combination of any of the above.

25. The method of claim 20, wherein: the seed culture medium comprises or consists of: 0 to 3 percent of glycerol, 0 to 1.5 percent of glucose, 0 to 1.5 percent of lactose, 0.2 to 2 percent of yeast organic nitrogen source, 0 to 2 percent of peptone, 0.1 to 2 percent of sodium chloride and 0 to 1 percent of sodium acetate.

26. The method of claim 24, wherein: the seed culture medium comprises or consists of: 1.2 to 1.8 percent of lactose, 0.4 to 0.8 percent of glucose, 0.6 percent of yeast extract powder, 1 percent of peptone, 2 percent of sodium chloride and 0 to 0.1 percent of defoaming agent.

27. The method of claim 20, wherein: the seed culture medium comprises a primary seed culture medium and a secondary seed culture medium, and the primary culture medium and the secondary culture medium can be the same or different;

the first-order seed culture medium comprises 1.2% of lactose, 0.4% of glucose, 0.6% of yeast extract powder, 1% of peptone and 2% of sodium chloride; the secondary seed culture medium comprises 1.8% of lactose, 0.8% of glucose, 0.5% of yeast extract powder, 1.5% of peptone, 2% of sodium chloride and 0.05% of defoaming agent.

28. The method of claim 26, wherein: in the first-stage seed culture process: the inoculation amount is more than or equal to 1 percent, the temperature is 25 to 34 ℃, and the rotating speed is more than or equal to 200rpm; the culture period is 20 to 54h;

in the secondary seed culture process: the inoculation amount is more than or equal to 0.03 percent; the temperature is 25 to 34 ℃; the pH value is more than or equal to 6.5; the culture period is 20 to 54h.

29. The method of claim 27, wherein: in the first-stage seed culture process: the inoculation amount is 4%, the temperature is 28 to 30 ℃, the rotating speed is 220 to 270rpm, and the culture period is 40 to 48h.

30. The method of claim 28, wherein: in the secondary seed culture process: the inoculation amount is 0.1 to 0.5 percent; the temperature is 28 to 30 ℃; the pH value is 6.8 to 7.8; the culture period is 20 to 48h.

31. The method of claim 3, wherein: the Tissuriella mobilis (A), (B) and (C)Tistrellamobilis) HDCC00053 (CGMCC NO. 22003) is inoculated to slant culture for activation before seed culture.

32. The method of claim 30, wherein: the slant culture medium is a combination of lactose, glycerol, glucose, yeast organic nitrogen sources, peptone, sodium chloride and agar.

33. The method of claim 31, wherein: the slant culture medium comprises lactose 0.4%, glycerol 0.4%, yeast extract powder 0.6%, peptone 1%, sodium chloride 2%, agar 1.8%, and pH7.0.