CN113913343B - Burkholderia thailand, application and fermentation method thereof - Google Patents
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Abstract
The invention provides a Burkholderia (Burkholderia) HDCC00029 which is preserved in China general microbiological culture Collection center (CGMCC) for a year 07 and a month 26 of 2021, wherein the preservation number is CGMCC NO.22963. The strain can be used for Thailand statin A fermentation production, the maximum fermentation titer can reach 4.07g/L, and the strain is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Burkholderia thailand, and an application and a fermentation method thereof.
Background
Telavastatin a (TST-a) has nano-to sub-nano-scale cytotoxicity to a variety of human cancer cell lines. Its mechanism of action is to bind tightly to the SF3b subunit of the U2 snRNA sub-complex, which is an important component of the spliceosome, thereby inhibiting spliceosome assembly. The present study has modified TST-A with N-hydroxysuccinimide (NHS) ester groups and coupled with trastuzumab via lysine amine groups, resulting in an almost "ligation-free" ADC. trastuzumab-TST has been tested in an in vitro HER2 overexpressing cell line and has been shown to accelerate the inhibition of splicing in N87 cells. In addition, trastuzumab-TST remains significantly potent against gastric cancer xenografts at doses as low as 1.5 mg/kg. In addition, TST-a based ADCs have a high therapeutic effect on the multidrug resistant (MDR) tumor phenotype compared to microtubule inhibitors such as maytansine or monomethyl oseltaine E (MMAE).
The researchers of the university of merisconsin, milwaki division, xiangyang Liu in 2012 found thailanthatin a, B, C from burkholderia thailand Burkholderia thailandensis MSMB. In 2016, thailand statin A was synthesized by organic synthetic chemists of university of Lesi, academy of sciences of the United states, K.C. Nicolaou, and research team, and the results of the related studies were published in J Journal of the American Chemical Society. Eustquio A S et al, biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp, reported that by genetically modifying the strain, the TST-A titer was increased from 0.347G/L to 2.5G/L by fermentation over 5 days using an optimized medium (tetracycline and L-arabinose were added to the 2S4G medium).
Disclosure of Invention
In order to obtain a strain with higher potency and suitable for industrialized production of telavancin A, the invention provides a strain of Burkholderia thailandBurkholderia thailandensis) HDCC00029 was preserved in the China general microbiological culture collection center (CGMCC) of the China general microbiological culture Collection center (CGMCC) for 26 months of 2021, and the preservation number is CGMCC No.22963.
The invention also provides an application of the Burkholderia striolata HDCC00029, which is used for preparing telavancin A (Thailanstatin A) and/or telavancin D (Thailanstatin D);
or, the preparation method is used for preparing one or more of medicines, cosmetics and feeds containing the telavastatin A and/or the telavastatin D.
The invention also discloses a fermentation broth containing the Burkholderia striolata HDCC 00029.
The invention also relates to the use of the fermentation broth for preparing telavastatin A and/or telavastatin D;
or, the preparation method is used for preparing one or more of medicines, cosmetics and feeds containing the telavastatin A and/or the telavastatin D.
The invention also provides a fermentation method for producing telavancin A and/or telavancin D by using the Burkholderia taenix HDCC 00029.
Preferably, the fermentation is carried out in a fermentation medium comprising an assimilable carbon source and/or nitrogen source.
Preferably, the assimilable carbon source is selected from one or a combination of any of starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol; preferably glucose, glycerol, sucrose, sorbitol, mannitol, maltose, lactose or any combination thereof; more preferably one of glucose, glycerol, sucrose or a combination of any of these.
Preferably, the assimilable nitrogen source is selected from one or a combination of any of an organic nitrogen source and an inorganic nitrogen source;
the organic nitrogen source is selected from one or a combination of more than one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cotton seed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal and peptone;
the inorganic nitrogen source is selected from one or a combination of any of urea and ammonium salt;
the assimilable nitrogen source is preferably soy peptone.
Preferably, the fermentation medium further comprises an inorganic salt selected from one or a combination of any of trisodium citrate, calcium carbonate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate; preferably one or a combination of any of calcium carbonate, ammonium sulfate and magnesium sulfate.
Preferably, the pH value of the fermentation medium is 5.0-8.8.
Advantageous effects
1. The strain provided by the invention is fermented and cultured in 30L culture medium for 5 days, and the titer of Thailand statin A can reach 4.07g/L;
2. the strain provided by the invention can be used for simultaneously fermenting and producing Thailand statin A and Thailand statin D;
3. the strain provided by the invention is suitable for industrial production.
Drawings
FIG. 1 shows an HPLC (high Performance liquid chromatography) spectrum of a shake flask fermentation broth of a TA11 strain, thailanthatin A (10.55 min), thailanthatin D (16.99 min);
FIG. 2 is a LCMS spectrum of TA11 strain shake flask fermentation broth Thailand A, m/z=536.2877 [ M+H ]] + , m/z =558.2688[M+Na] + ；
FIG. 3 is a LCMS spectrum of TA11 strain shake flask fermentation broth Thailand D, m/z=520.2923 [ M+H ]] + , m/z =542.2744[M+Na] + ；
FIG. 4-A is a photograph of streaked colonies on a nutrient agar plate, and FIG. 4-B is a photomicrograph of a seed culture;
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the following examples are commercially available products unless otherwise specified.
The invention will be further described by way of the following examples, which are not intended to limit the scope of the invention in any way. It will be appreciated by those skilled in the art that equivalent substitutions and modifications may be made to the teachings of the present invention, and that such modifications may still fall within the scope of the present invention.
In the following examples, the HPLC detection method is:
chromatographic column: agilent POROSHELL EC-C18 4.6100mm 2.7um
Column temperature: 40 DEG C
Sample injection amount: 5ul
Mobile phase: a (0.1% formic acid in water): b (0.1% acetonitrile formate solution) =55: 45
Flow rate: 1.0ml/min.
EXAMPLE 1 isolation and screening of original Producer Strain
Noni stem blocks in tropical areas (three-in-sea) of China are taken and put into 50mL of sterile water, and are oscillated at 250rpm for 10 min to obtain tissue surface bacteria stock solution. The washed noni stem block is chopped and put into 50mL of sterile water, and the noni stem tissue internal bacterial stock solution is obtained after shaking for 10 min at 250 rpm. Enrichment culturing the two bacterial stock solutions at 35 ℃ for 16 hours, and sequentially diluting the two bacterial stock solutions to 10 respectively by using sterile water -2 、10 -3 、10 -4 、10 -5 Four bacterial solutions with different concentrations are prepared by coating 100 mu L of each bacterial solution on a nutrient agar plate, inverted and dried, and then placed in a 35 ℃ incubator for culture. After the colony grows out, selecting a plate with single colony coated with proper concentration, picking round, smooth and wet single colony from the plate, respectively scribing, separating and purifying on a nutrient agar plate, and preserving the purified strain by using a nutrient agar test tube inclined plane. And (3) respectively carrying out 16S rDNA sequence determination on the preserved strains, selecting strains with high homology with Burkholderia, further carrying out rescreening by adopting shaking flask fermentation, detecting the rescreened fermentation liquor by adopting HPLC, retaining the strains and samples with the same retention time as Thailand A reference substances, further carrying out molecular weight determination by adopting LCMS, and finally successfully obtaining the original production strains (number TA 11) for simultaneously producing Thailand A and Thailand D. HPLC patterns of strain TA11 shake flask fermentation broth are shown in FIG. 1, and fractions at 10.55min and 16.99min are collected, and LCMS patterns are shown in FIG. 2 and FIG. 3, respectively.
Example 2
As shown in FIGS. 4-A and 4-B, the strain (original number TA 11) was streaked onto a nutrient agar plate, and incubated at 35℃for 24 hours, and the colony was pale yellow, round, smooth and moist, slightly convex, translucent, and clean-edged. The strain is in a single rod shape, better in dyeing and less in spores when observed under a microscope.
The physiological and biochemical identification is characterized by liquefied gelatin, reduced nitrate, V.P test positive, hydrolyzed starch and lecithin enzyme test negative.
The 16S rDNA sequence measured by the strain is compared with the sequences of related species and genus in the GenBank database by BLAST of homologous sequences after being checked, and the strain is finally determined to be Burkholderia of Thailand by combining the physiological and biochemical test resultsBurkholderia thailandensis) The 16S rDNA sequence of the strain TA11 is shown in SEQ ID NO. 1.
Through culture and screening, the carbon source is optimally utilized by glucose, glycerol and sucrose, and the utilization capacity of the dextrin, starch and xylose is poorer than that of sorbitol, mannitol, maltose and lactose. As the nitrogen source, a plurality of organic nitrogen sources such as yeast nitrogen source, peptone and corn steep liquor, and conventional inorganic nitrogen sources such as ammonium salt, urea and nitrate can be used. The pH test result shows that the optimal growth pH range is 5.0-8.8.
EXAMPLE 3 bacterial mutagenesis preliminary screening
Inoculating 0.1ml of glycerol tube with nutrient agar slant by using original strain (TA 11) as original strain, culturing overnight at 35deg.C, washing fresh lawn with sterile physiological saline, adding glass beads, shaking, and scattering to obtain bacterial suspension. The bacterial suspension is mixed with 1200 mug/ml EMS mother liquor in equal volume and placed on a shaking table at 35 ℃ for mutagenesis treatment for 30min. The mutagenic solution was centrifuged at 14000rpm in a centrifuge tube, the supernatant was discarded, and then resuspended in 0.1% (w/v) sodium thiosulfate solution, and the solution was repeatedly centrifuged and washed 3 times to perform gradient dilution. The dilutions with different dilution gradients are coated on a nutrient agar plate and placed at 35 ℃ for culture for 24 hours, so that isolated single colonies are obtained. Separating single colony to seed on fresh nutrient agar plate, culturing at 35deg.C for 16 hr to obtain purified seed single colony, scraping small amount of thallus from each colony with inoculating shovel, inoculating into liquid primary screening fermentation medium, stirring, and shake culturing at 30deg.C on 220rpm shaker for 6 days to obtain primary screening fermentation liquid. 1ml of fermentation broth is taken, soaked in 95% (v/v) ethanol and subjected to ultrasonic treatment for 30min, and after centrifugal filtration, HPLC detection is carried out. In this case 2437 single colonies were selected, with the highest production of Thailand statin A, up to 2.46g/L.
The primary screening fermentation medium is a 2S4G medium, and the composition of the primary screening fermentation medium is as follows: 40 g/L glycerol, 12.5. 12.5 g/L soytone, 2. 2g/L (NH) 4 ) 2 SO 4 ，0.1 g/L MgSO 4 ·7H 2 O，2 g/L CaCO 3 ，pH 7.0。
EXAMPLE 4 Compound Screen purification of seed
Taking a TA-NS-863 strain glycerol tube, inoculating a nutrient agar plate by a streaking method, culturing at 35 ℃ overnight to obtain single colonies, scraping a small amount of thalli from each colony by an inoculating shovel, inoculating to a liquid LB seed culture medium, and culturing at 35 ℃ for 8 hours to obtain seed liquid. Inoculating the seed solution into a double-sieve fermentation medium at a ratio of 5%, and placing the double-sieve fermentation medium on a shaking table at 30 ℃ and 220rpm for shaking culture for 6 days to obtain the double-sieve fermentation liquid. 1ml of fermentation broth is taken, soaked in 95% (v/v) ethanol and subjected to ultrasonic treatment for 30min, and after centrifugal filtration, HPLC detection is carried out. In the case, 20 single colonies are checked, the yield of Thailand statin A is 2.42-2.49 g/L, and the average yield is 2.47g/L. Taking the colony with the highest yield (2.49 g/L), inoculating nutrient agar inclined plane for expansion culture, and preserving the strain as Thailand statin A high-yield original strain (with the number of HDCC 00029), wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 22963 in the year 07 of 2021 and 26.
The re-screening fermentation medium is a 2S4G medium, and comprises the following components: 40 g/L glycerol, 12.5. 12.5 g/L soytone, 2. 2g/L (NH) 4 ) 2 SO 4 ，0.1 g/L MgSO 4 ·7H 2 O，2 g/L CaCO 3 ，pH 7.0。
Example 5 shake flask fermentation test
Inoculating a nutrient agar plate by a streaking method to a glycerol tube of a CGMCC No.22963 strain, culturing overnight at 35 ℃ to obtain single colonies, scraping a small amount of thalli by an inoculating shovel, inoculating to a liquid LB seed culture medium, and culturing at 35 ℃ for 8 hours to obtain seed liquid. Inoculating the seed solution into a fermentation medium at a ratio of 5%, placing the fermentation medium on a shaking table at 25 ℃, and carrying out shaking culture at 220rpm for 6 days to obtain a fermentation liquid. 1ml of fermentation broth is taken, soaked in 95% (v/v) ethanol for 30min, centrifugally filtered and subjected to HPLC detection, wherein the content of Thailand statin A in the fermentation broth is 2.52g/L, and the content of Thailand statin D in the fermentation broth is 0.22 g/L.
The fermentation medium is a 2S4G medium, and the composition of the fermentation medium is as follows: 40 g/L glycerol, 12.5 g/L soytone, 2g/L (NH 4) 2SO4,0.1 g/L MgSO4.7H2O, 2g/L CaCO3, pH 7.0.
EXAMPLE 6 30L fermentation test
Inoculating a nutrient agar plate by a streaking method to a glycerol tube of a CGMCC No.22963 strain, culturing overnight at 35 ℃ to obtain single colonies, scraping a small amount of thalli by an inoculating shovel, inoculating to a liquid LB seed culture medium, and culturing at 35 ℃ for 8 hours to obtain a first-stage shake flask seed liquid. Inoculating the first-stage shake flask seed liquid into a seed tank with 10L of LB liquid culture medium at 0.05%, setting the temperature to 35 ℃, the tank pressure to 0.05Mpa, the air flow to 1vvm, initially stirring at 100rpm, controlling dissolved oxygen to be more than or equal to 40% in a stirring linkage manner, and culturing for 7h to obtain the second-stage seed liquid. Inoculating the second-stage seed liquid into a fermentation tank filled with 30L of liquid fermentation medium according to the proportion of 5%, setting the temperature to 25 ℃, setting the tank pressure to 0.05Mpa, setting the air flow to 0.7vvm, initially stirring at 80rpm, starting stirring linkage when the dissolved oxygen is reduced to below 15%, and controlling the dissolved oxygen to be more than or equal to 30%. When the glycerol is reduced to below 2% (w/v), feeding is started, the feeding speed is regulated according to the concentration of the glycerol in the fermentation liquid, the concentration of the glycerol is controlled to be 2-3%, and the fermentation period is 5 days. 1ml of fermentation broth is taken, soaked in 95% (v/v) ethanol for 30min, subjected to centrifugal filtration and subjected to HPLC detection, wherein the content of Thailand statin A in the fermentation broth is 4.07g/L, and the content of Thailand statin D in the fermentation broth is 0.34g/L.
The fermentation medium is a 2S4G medium, and the composition of the fermentation medium is as follows: 40 g/L glycerol, 12.5. 12.5 g/L soytone, 2. 2g/L (NH) 4 ) 2 SO 4 ，0.1 g/L MgSO 4 •7H 2 O，2 g/L CaCO 3 ，pH 7.0。
The feed medium consists of: glycerol 40% (w/v), ammonium sulfate 5% (w/v).
Sequence listing
<110> Zhejiang to Biotechnology Co., ltd
<120> Burkholderia thailand strain, application and fermentation method thereof
<130> P0102021110838
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1490
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
agattgaacg ctggcggcat gggttacaca tgcaagtcga acggcagcgc gggcttcggc 60
ctggcggcga gtggcgaacg ggtgagtaat acatcggaac atgtcctgta gtgggggata 120
gcccggcgaa agccggatta ataccgcata cgatctgtgg atgaaagcgg gggaccttcg 180
ggcctcgcgc tatagggttg gccgatggct gattagctag ttggtggggt aaaggcctac 240
caaggcgacg atcagtagct ggtctgagag gacgaccagc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtggg gaattttgga caatgggcgc aagcctgatc 360
cagcaatgcc gcgtgtgtga agaaggcctt cgggttgtaa agcacttttg tccggaaaga 420
aatcatcctg gctaataacc ggggtggatg acgagcccgg aagaataagc accggctaac 480
tacgtgccag cagccgcggt aatacgtagg gtgcgagcgt taatcggaat tactgggcgt 540
aaagcgtgcg caggcggttt gctaagaccg atgtgaaatc cccgggctca acctgggaac 600
tgcattggtg actggcaggc tagagtatgg cagagggggg tagaattcca cgtgtagcag 660
tgaaatgcgt agagatgtgg aggaataccg atggcagcgg cagccccctg ggccaatact 720
gacgctcatg cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780
ctaaacgatg tcaactagtt gttggggatt catttcctta gtaacgtagc taacgcgtga 840
agttgaccgc ctggggagta cggtcgcaag attaaaactc aaaggaattg acggggaccc 900
gcacaagcgg tggatgatgt ggattaattc gatgcaacgc gaaaaacctt acctaccctt 960
gacatggtcg gaatcctgct gagaggcggg agtgctcgaa agagaaccgg cgcacaggta 1020
ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080
acccttgtcc ttagttgcta cgcaagagca ctctaaggag actgccggtg acaaaccgga 1140
agaaggtggg gatgacgtca agtcctcatg gcccttatgg gtagggcttc acacgtcata 1200
caatggtcgg aacagagggt cgccaacccg cgagggggag ccaatcccag aaaaccgatc 1260
gtagtccgga ttgcactctg caactcgagt gcatgaagct ggaatcgcta gtaatcgcgg 1320
atcagcatgc cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccatgg 1380
gagtgggttt taccagaagt ggctagtcta accgcaagga ggacggtcac cacggtagga 1440
ttcatgactg gggtgaagtc gtaacaaggt agccgtatcg gaaggtgctc 1490
Claims (14)
1. Burkholderia strainBurkholderia thailandensis) HDCC00029 was deposited in the China general microbiological culture collection center, with the accession number of CGMCC No.22963, month 07, 2021, and 26.
2. Use of burkholderia HDCC00029 according to claim 1, characterized in that:
for the preparation of telavastatin a and/or telavastatin D;
or, the preparation method is used for preparing one or more of medicines, cosmetics and feeds containing the telavastatin A and/or the telavastatin D.
3. A fermentation broth comprising burkholderia striolata HDCC00029 according to claim 1.
4. A use of the fermentation broth of claim 3, wherein:
for the preparation of telavastatin a and/or telavastatin D;
or, the preparation method is used for preparing one or more of medicines, cosmetics and feeds containing the telavastatin A and/or the telavastatin D.
5. A fermentation process for the production of telavastatin a and/or telavastatin D using burkholderia HDCC00029 according to claim 1.
6. The fermentation process of claim 5, wherein:
comprising carrying out the fermentation in a fermentation medium comprising an assimilable carbon source and/or nitrogen source.
7. The fermentation process of claim 6, wherein:
the assimilable carbon source is selected from one or a combination of any of starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol and mannitol.
8. The fermentation process of claim 6, wherein:
the assimilable carbon source is selected from one or a combination of several of glucose, glycerol, sucrose, sorbitol, mannitol, maltose and lactose.
9. The fermentation process of claim 6, wherein:
the assimilable carbon source is selected from one or a combination of several of glucose, glycerol and sucrose.
10. The fermentation process of claim 6, wherein:
the assimilable nitrogen source is selected from an organic nitrogen source and/or an inorganic nitrogen source;
the organic nitrogen source is selected from one or a combination of more than one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cotton seed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal and peptone; the inorganic nitrogen source is selected from urea and/or ammonium salts.
11. The fermentation process of claim 6, wherein:
the assimilable nitrogen source is soybean peptone.
12. The fermentation process of claim 6, wherein:
the fermentation medium further comprises inorganic salt, wherein the inorganic salt is selected from one or a combination of several of trisodium citrate, calcium carbonate, 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.
13. The fermentation process of claim 6, wherein:
the fermentation medium further comprises an inorganic salt selected from one or a combination of any of calcium carbonate, ammonium sulfate and magnesium sulfate.
14. The fermentation method according to any one of claims 6 to 13, wherein:
the pH value of the fermentation medium is 5.0-8.8.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111788208A (en) * | 2017-09-20 | 2020-10-16 | Ph制药有限公司 | Talanstatin analogs |
| CN112746036A (en) * | 2020-12-08 | 2021-05-04 | 浙江珲达生物科技有限公司 | Streptomyces and method for producing pseudouridine by fermenting same |
| CN113564071A (en) * | 2021-07-16 | 2021-10-29 | 浙江珲达生物科技有限公司 | Bacillus natto for producing menadione-7 and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111788208A (en) * | 2017-09-20 | 2020-10-16 | Ph制药有限公司 | Talanstatin analogs |
| CN112746036A (en) * | 2020-12-08 | 2021-05-04 | 浙江珲达生物科技有限公司 | Streptomyces and method for producing pseudouridine by fermenting same |
| CN113564071A (en) * | 2021-07-16 | 2021-10-29 | 浙江珲达生物科技有限公司 | Bacillus natto for producing menadione-7 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation;Xiangyang Liu等;《Synth Syst Biotechnol》;第1卷(第1期);34-38 * |
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