CN110157752A - The culture medium of natural products is prepared for Burkholderia mesh DSM7029 strain fermentation - Google Patents
The culture medium of natural products is prepared for Burkholderia mesh DSM7029 strain fermentation Download PDFInfo
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- CN110157752A CN110157752A CN201810149000.6A CN201810149000A CN110157752A CN 110157752 A CN110157752 A CN 110157752A CN 201810149000 A CN201810149000 A CN 201810149000A CN 110157752 A CN110157752 A CN 110157752A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 85
- 230000004151 fermentation Effects 0.000 title claims abstract description 85
- 229930014626 natural product Natural products 0.000 title claims abstract description 39
- 241001453380 Burkholderia Species 0.000 title claims abstract description 32
- 239000001963 growth medium Substances 0.000 title claims description 68
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 135
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 68
- 229930006000 Sucrose Natural products 0.000 claims abstract description 68
- 239000005720 sucrose Substances 0.000 claims abstract description 66
- 235000011187 glycerol Nutrition 0.000 claims abstract description 45
- 229930013356 epothilone Natural products 0.000 claims abstract description 36
- 229920005989 resin Polymers 0.000 claims abstract description 32
- 239000011347 resin Substances 0.000 claims abstract description 32
- 239000011573 trace mineral Substances 0.000 claims abstract description 32
- 235000013619 trace mineral Nutrition 0.000 claims abstract description 32
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 31
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims abstract description 31
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims abstract description 31
- 239000000843 powder Substances 0.000 claims abstract description 31
- 108010009004 proteose-peptone Proteins 0.000 claims abstract description 31
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 30
- 229920001353 Dextrin Polymers 0.000 claims abstract description 19
- 239000004375 Dextrin Substances 0.000 claims abstract description 19
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 19
- 235000019425 dextrin Nutrition 0.000 claims abstract description 19
- ZIYVHBGGAOATLY-UHFFFAOYSA-N methylmalonic acid Chemical compound OC(=O)C(C)C(O)=O ZIYVHBGGAOATLY-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000001632 sodium acetate Substances 0.000 claims abstract description 19
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 19
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 claims abstract description 19
- 239000004324 sodium propionate Substances 0.000 claims abstract description 19
- 229960003212 sodium propionate Drugs 0.000 claims abstract description 19
- 235000010334 sodium propionate Nutrition 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 15
- 235000001727 glucose Nutrition 0.000 claims abstract description 15
- 239000008103 glucose Substances 0.000 claims abstract description 15
- 229920002472 Starch Polymers 0.000 claims abstract description 14
- 239000008107 starch Substances 0.000 claims abstract description 14
- 235000019698 starch Nutrition 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 6
- 229960005150 glycerol Drugs 0.000 claims abstract description 5
- 229960004249 sodium acetate Drugs 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 229960004793 sucrose Drugs 0.000 claims description 65
- 239000002609 medium Substances 0.000 claims description 53
- 150000003883 epothilone derivatives Chemical class 0.000 claims description 29
- 238000001179 sorption measurement Methods 0.000 claims description 22
- 229920001429 chelating resin Polymers 0.000 claims description 14
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 13
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 13
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 13
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 9
- 208000035404 Autolysis Diseases 0.000 claims description 8
- 206010057248 Cell death Diseases 0.000 claims description 8
- 108091008053 gene clusters Proteins 0.000 claims description 8
- 230000028043 self proteolysis Effects 0.000 claims description 8
- 108010030975 Polyketide Synthases Proteins 0.000 claims description 3
- 108010000785 non-ribosomal peptide synthase Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 241000862998 Polyangium Species 0.000 claims description 2
- 108010019477 S-adenosyl-L-methionine-dependent N-methyltransferase Proteins 0.000 claims description 2
- 239000003463 adsorbent Substances 0.000 claims 4
- 241000186146 Brevibacterium Species 0.000 claims 1
- HESCAJZNRMSMJG-KKQRBIROSA-N epothilone A Chemical class C/C([C@@H]1C[C@@H]2O[C@@H]2CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 HESCAJZNRMSMJG-KKQRBIROSA-N 0.000 abstract description 6
- -1 Epothilones Natural products 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract description 2
- 239000002250 absorbent Substances 0.000 abstract 1
- 230000002745 absorbent Effects 0.000 abstract 1
- 239000000654 additive Substances 0.000 abstract 1
- 230000000996 additive effect Effects 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000007788 liquid Substances 0.000 description 12
- 238000009630 liquid culture Methods 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 230000008859 change Effects 0.000 description 9
- 241000987691 [Polyangium] brachysporum Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000012935 Averaging Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002386 leaching Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- CXQWRCVTCMQVQX-LSDHHAIUSA-N (+)-taxifolin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC=C(O)C(O)=C1 CXQWRCVTCMQVQX-LSDHHAIUSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- XOZIUKBZLSUILX-UHFFFAOYSA-N desoxyepothilone B Natural products O1C(=O)CC(O)C(C)(C)C(=O)C(C)C(O)C(C)CCCC(C)=CCC1C(C)=CC1=CSC(C)=N1 XOZIUKBZLSUILX-UHFFFAOYSA-N 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 101000979117 Curvularia clavata Nonribosomal peptide synthetase Proteins 0.000 description 1
- BEFZAMRWPCMWFJ-JRBBLYSQSA-N Epothilone C Natural products O=C1[C@H](C)[C@@H](O)[C@@H](C)CCC/C=C\C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C BEFZAMRWPCMWFJ-JRBBLYSQSA-N 0.000 description 1
- XOZIUKBZLSUILX-SDMHVBBESA-N Epothilone D Natural products O=C1[C@H](C)[C@@H](O)[C@@H](C)CCC/C(/C)=C/C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C XOZIUKBZLSUILX-SDMHVBBESA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- BEFZAMRWPCMWFJ-UHFFFAOYSA-N desoxyepothilone A Natural products O1C(=O)CC(O)C(C)(C)C(=O)C(C)C(O)C(C)CCCC=CCC1C(C)=CC1=CSC(C)=N1 BEFZAMRWPCMWFJ-UHFFFAOYSA-N 0.000 description 1
- KQNGHARGJDXHKF-UHFFFAOYSA-N dihydrotamarixetin Natural products C1=C(O)C(OC)=CC=C1C1C(O)C(=O)C2=C(O)C=C(O)C=C2O1 KQNGHARGJDXHKF-UHFFFAOYSA-N 0.000 description 1
- BEFZAMRWPCMWFJ-QJKGZULSSA-N epothilone C Chemical compound O1C(=O)C[C@H](O)C(C)(C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)CCC\C=C/C[C@H]1C(\C)=C\C1=CSC(C)=N1 BEFZAMRWPCMWFJ-QJKGZULSSA-N 0.000 description 1
- XOZIUKBZLSUILX-GIQCAXHBSA-N epothilone D Chemical compound O1C(=O)C[C@H](O)C(C)(C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)CCC\C(C)=C/C[C@H]1C(\C)=C\C1=CSC(C)=N1 XOZIUKBZLSUILX-GIQCAXHBSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000029115 microtubule polymerization Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229960003552 other antineoplastic agent in atc Drugs 0.000 description 1
- 229920001470 polyketone Polymers 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of culture mediums that natural products is prepared for 7029 strain fermentation of Burkholderia mesh DSM, in addition to comprising casein peptone, yeast powder, magnesium chloride hexahydrate, glycerol, sodium acetate, sodium propionate, methylmalonic acid, trace element solution and macroporous absorbent resin, the also carbon source component comprising additionally adding, carbon source group is selected from one or more of sucrose, starch, dextrin, glucose and glycerol, and preferred additive amount is that sucrose 5-50g/L or starch 5-50g/L or dextrin 5-50g/L or glycerol 5-50mL/L or sucrose add glucose 5-50g/L.It is experimentally confirmed that this composition and optimization significantly improve the fermentation level of the natural products such as Epothilones, carrying out that there is applications well prospect when fermentation prepares natural products using 7029 bacterial strain of Burkholderia mesh DSM.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium for preparing a natural product by fermenting a strain DSM7029 of Burkholderia.
Background
Burkholderia [ Polyangium ] brachysporum DSM7029 (═ K481-B101 ═ ATCC 53080) was isolated from soil in Greece in 1988. The growth speed of the strain is high, single colony can be seen in two days, and the genetic operation is simple and convenient. The DSM7029 strain produces the antifungal and antitumor drug taxicin, a natural product of the hybrid NRPS/PKS (non-ribosomal peptide synthetase/polyketide synthase) type.
The DSM7029 strain can also be used for heterogeneously expressing a novel antitumor drug epothilone. The epothilone compound belongs to a macrolide compound of polyketone, can stabilize microtubule polymerization, and has an action mechanism similar to that of taxol. More importantly, epothilones also have good activity against cancer cells that are ineffective against paclitaxel and other antineoplastic agents. The epothilone is heterogeneously expressed in DSM7029 strain, and the yield can reach 307 mug/L after modification. However, the fermentation yield is a considerable distance from the requirements of industrial production. At present, the related literature reports on the composition and optimization of the culture medium for preparing the natural product by fermenting the strain DSM7029 belonging to Burkholderia are deficient, so that how to obtain the culture medium capable of effectively improving the yield of the natural product prepared by fermenting the strain DSM7029 belonging to Burkholderia becomes an urgent problem to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a culture medium for preparing a natural product by fermenting the strain DSM7029 belonging to Burkholderia, so that the fermentation yield of the natural product is effectively improved, and the autolysis phenomenon in the fermentation process of thalli is improved.
The specific technical scheme of the invention is as follows:
the invention provides a culture medium for preparing natural products by fermentation of a Burkholderia DSM7029 strain, which comprises casein peptone, yeast powder, magnesium chloride hexahydrate, glycerol, sodium acetate, sodium propionate, methylmalonic acid, a trace element solution and macroporous adsorption resin, and also comprises an additional carbon source component which is selected from one or more of sucrose, starch, dextrin, glucose and glycerol.
Preferably, the components of the trace element solution at least comprise manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate and ferrous sulfate heptahydrate.
Preferably, the macroporous adsorption resin is amberlite XAD-16 macroporous adsorption resin.
Preferably, sucrose is added in an amount of 5 to 50g/L in the carbon source component additionally added.
Preferably, the starch is added in an amount of 5-50g/L in the additionally added carbon source component.
Preferably, in the additionally added carbon source component, the addition amount of dextrin is 5-50 g/L.
Preferably, glycerol is added in an amount of 5-50mL/L in the carbon source component additionally added.
Preferably, sucrose and glucose are added in an amount of 5-50g/L in the additionally added combined carbon source component.
More preferably, sucrose is added in an amount of 25g/L in the carbon source component additionally added.
More preferably, in the additionally added carbon source component, the added amount of starch is 25 g/L.
More preferably, in the additionally added carbon source component, the addition amount of dextrin is 25 g/L.
More preferably, glycerol is added in an amount of 25mL/L in the carbon source component additionally added.
More preferably, in the carbon source component additionally added, the addition amount of sucrose is 5g/L and the addition amount of glucose is 10 g/L.
In a preferred embodiment, the content of the casein peptone is 8g/L, the content of the yeast powder is 4g/L, the content of the magnesium chloride hexahydrate is 4.06g/L, the content of the non-additionally added glycerol is 5mL/L, the content of the sodium acetate is 50mg/L, the content of the sodium propionate is 100mg/L, the content of the methylmalonic acid is 100mg/L, the content of the trace element solution is 1mL/L, and the content of the macroporous adsorption resin is 20 mL/L.
Preferably, in the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
In a second aspect, the invention provides a culture medium for improving autolysis of a burkholderia DSM7029 strain in a bacterial fermentation process, wherein the culture medium comprises casein peptone, yeast powder, magnesium chloride hexahydrate, glycerol, sodium acetate, sodium propionate, methylmalonic acid, a trace element solution and macroporous adsorption resin, and additionally added sucrose.
Preferably, the components of the trace element solution at least comprise manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate and ferrous sulfate heptahydrate.
Preferably, the macroporous adsorption resin is amberlite XAD-16 macroporous adsorption resin.
Preferably, the amount of sucrose added is 5-50 g/L.
More preferably, sucrose is added in an amount of 25 g/L.
In a preferred embodiment, the content of the casein peptone is 8g/L, the content of the yeast powder is 4g/L, the content of the magnesium chloride hexahydrate is 4.06g/L, the content of the non-additionally added glycerol is 5mL/L, the content of the sodium acetate is 50mg/L, the content of the sodium propionate is 100mg/L, the content of the methylmalonic acid is 100mg/L, the content of the trace element solution is 1mL/L, and the content of the macroporous adsorption resin is 20 mL/L.
Preferably, in the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
In a third aspect, the invention provides the use of a medium as described above in the preparation of a natural product by fermentation, the Burkholderia DSM7029 strain being [ Polyangium ] brachyspora DSM 7029.
In a preferred embodiment, the natural product is a product of a gene cluster consisting of polyketide synthase and/or non-ribosomal peptide synthase and derivatives thereof.
More preferably, the natural products include at least epothilones and taxicins.
In a fourth aspect, the present invention provides a method for increasing the yield of a natural product produced by fermentation of a strain of Burkholderia DSM7029, which method uses the medium provided in the first aspect of the invention as the fermentation medium for the strain of Burkholderia DSM 7029.
In a fifth aspect, the invention provides a method for improving autolysis of the Burkholderia DSM7029 strain in a bacterial fermentation process, wherein the method adopts the culture medium provided by the second aspect of the invention as a fermentation medium of the Burkholderia DSM7029 strain.
The invention provides a culture medium for preparing a natural product by fermenting a strain DSM7029 of Burkholderia, and the fermentation level of the natural product is obviously improved by adding components such as sucrose, starch, dextrin, glucose, glycerol and the like into the culture medium. The invention has the beneficial effects that: the added carbon source components are cheap and easy to obtain, and the preparation is simple; compared with the result of a contrast culture medium, the yield is obviously improved and reaches 28mg/L at most, the industrial requirement of preparing a natural product by fermenting the Burkholderia DSM7029 strain is basically met, and the method has good application prospect.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space. It is therefore contemplated to cover by the present invention, equivalents and modifications that fall within the scope of the invention, and that fall within the scope of the invention.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows the results of comparison of the yields of natural products when different carbon sources are added to the medium in a preferred embodiment of the invention;
FIG. 2 shows the effect of different concentrations of sucrose on the yield of epothilone fermentation by strain MMR11 in a preferred embodiment of the invention;
FIG. 3 shows the effect of different concentrations of dextrin on the yield of epothilone fermentation by strain MMR11 in a preferred embodiment of the invention;
FIG. 4 shows the observation results of the cell morphology of 6 days after adding sucrose to the MMR11 culture medium of the strain to ferment epothilone in the preferred embodiment of the present invention;
FIG. 5 shows the change of viable count of 6 days after adding sucrose to the culture medium of strain MMR11 to ferment epothilone in a preferred embodiment of the invention;
FIG. 6 shows the change in OD600 of 6 days after fermentation of epothilone by addition of sucrose to strain MMR11 medium in a preferred embodiment of the invention;
FIG. 7 shows the change in pH of the culture medium of strain MMR11 after 6 days of sucrose addition for epothilone fermentation in a preferred embodiment of the invention;
FIG. 8 shows the change in sucrose concentration of strain MMR11 medium after 6 days of sucrose addition for epothilone fermentation in a preferred embodiment of the present invention;
FIG. 9 shows the effect of sucrose addition to the medium on the yield (peak area) of taxicin in a preferred embodiment of the invention;
FIG. 10 shows the effect of sucrose and glucose addition to the culture medium of strain MMR11 on epothilone production in a preferred embodiment of the invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL/L, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight:
control medium: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
Medium with additional sucrose addition: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 15g/L of sucrose, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
Medium with additional addition of starch: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 15g/L of starch, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
Medium with additional addition of dextrin: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 15g/L of dextrin, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
Medium with additional glycerol addition: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL/L, glycerol (added additionally) 15mL/L, sodium acetate 50mg/L, sodium propionate 100mg/L, methylmalonic acid 100mg/L, trace element solution 1mL/L, amberlite XAD-16 macroporous adsorption resin 20 mL/L.
In the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
4. Separation, extraction, purification and detection of epothilone: after 6 days of fermentation, the resin was collected by filtration through a 100 mesh screen and extracted twice overnight in a triangular flask with the addition of 25mL of methanol. After being combined, 2mL of leaching liquor is taken, filtered by a 0.22 mu m filter and detected by mass spectrum.
5. And (4) calculating the yield of the natural product according to a curve of the relation between the absorption peak area and the concentration of the standard sample, and averaging the three groups of results and calculating the error.
Compared with a culture medium without adding a carbon source, the culture medium containing the culture medium added with the sucrose, the starch, the dextrin or the glycerol has the advantage that the yield of natural products is obviously improved. Specifically, the differences in natural product production are shown in FIG. 1. The results show that when the strain DSM7029 of Burkholderia is fermented to prepare a natural product, the fermentation yield of the natural product is obviously improved by using a culture medium added with cane sugar, starch, dextrin or glycerol (epoC: epothilone C; epoD: epothilone D). The total amount of natural product was increased by about 9.6 times when sucrose was added compared to the control medium; the total amount of natural product was increased by about 5.2 times when starch was added compared to the control medium; the total amount of natural product was increased by about 11-fold with dextrin addition compared to the control medium; the total amount of natural product was increased by about 3.2-fold when glycerol was added compared to the control medium.
Example 2
Experiment for optimizing addition amount of sucrose
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 0-35g/L of sucrose, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
In the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
4. Separation, extraction, purification and detection of epothilone: after 6 days of fermentation, the resin was collected by filtration through a 100 mesh screen and extracted twice overnight in a triangular flask with the addition of 25mL of methanol. After being combined, 2mL of leaching liquor is taken, filtered by a 0.22 mu m filter and detected by mass spectrum.
5. And (4) calculating the yield of the natural product according to a curve of the relation between the absorption peak area and the concentration of the standard sample, and averaging the three groups of results and calculating the error.
The production of epothilones was examined as shown in FIG. 2. The results show that the addition of different concentrations of sucrose to the fermentation medium increases the yield of epothilone. Sucrose 25g/L is the most preferred addition level, in combination with natural product yield and cost considerations.
Example 3
Experiment for optimizing addition amount of dextrin
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 0-50g/L of dextrin, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
4. Separation, extraction, purification and detection of epothilone: after 6 days of fermentation, the resin was collected by filtration through a 100 mesh screen and extracted twice overnight in a triangular flask with the addition of 25mL of methanol. After being combined, 2mL of leaching liquor is taken, filtered by a 0.22 mu m filter and detected by mass spectrum.
5. And (4) calculating the yield of the natural product according to a curve of the relation between the absorption peak area and the concentration of the standard sample, and averaging the three groups of results and calculating the error.
The production of epothilones was examined as shown in FIG. 3. The results show that the addition of different concentrations of dextrin to the fermentation medium increases the yield of epothilone. Dextrin 25g/L is the most preferred amount to add, in combination with natural product yield and cost considerations.
Example 4
The autolysis phenomenon in the thallus fermentation process can be improved after the sucrose is added
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 0 or 25g/L of sucrose, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
In the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
4. Fermentation parameter monitoring: and (3) taking fermentation liquor without adding sucrose or with adding 25g/L of sucrose after 24, 48, 72, 96, 120 and 144 hours of fermentation, observing the shape change of the thallus by using a frozen scanning electron microscope, and detecting the viable count, OD600, pH and sucrose concentration. FIG. 4 shows the observation of the cell morphology of 6 days after sucrose addition fermentation of epothilone by strain MMR11 medium, showing that the cell was almost completely autolyzed by the third day of fermentation when fermentation was performed without sucrose addition; after the addition of sucrose, autolysis did not occur from the fifth day of fermentation, and non-autolysed mycelia remained until the sixth day. The sucrose is added in the fermentation process, so that the autolysis phenomenon of DSM7029 can be effectively improved. FIG. 5 shows the change in viable count of 6 days for sucrose-supplemented epothilone fermentation by strain MMR11, showing that viable count decreased significantly from the third day of fermentation when fermentation was performed without sucrose supplementation; when sucrose was added, there was still more viable bacteria until the sixth day. The addition of sucrose in the fermentation process can effectively maintain the number of viable bacteria. FIG. 6 shows the change in OD600 of the strain MMR11 medium after sucrose addition for 6 days of epothilone fermentation, showing that the cell density decreased from the second day of fermentation without a stable plateau when fermentation was performed without sucrose addition; when sucrose is added, the thallus density is kept stable within four days, and a stable plateau period is provided. The addition of sucrose in the fermentation process can effectively maintain the thallus density. FIG. 7 change in pH of the MMR11 medium after 6 days of sucrose addition for epothilone fermentation, showing that the pH increased from the second day of fermentation when fermentation was performed without sucrose addition; after sucrose addition, it remained neutral until the sixth day. The pH value in the fermentation process can be effectively stabilized by adding the sucrose in the fermentation process. FIG. 8 shows the change of sucrose concentration in the culture medium MMR11 of strain after 6 days of sucrose addition for epothilone fermentation, and the results show that sucrose is utilized in the fermentation process of the strain.
Example 6
Addition of sucrose Medium to ferment taxi rhzomorphs using DSM7029
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 0 or 25g/L of sucrose, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
In the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
4. Separation, extraction, purification and detection of taxifolin: after 6 days of fermentation, the resin was collected by filtration through a 100 mesh screen and extracted twice overnight in a triangular flask with the addition of 25mL of methanol. Mixing, collecting 2mL of the extract, filtering with 0.22 μm filter, and detecting with high performance liquid chromatography at 249 nm.
The production of taxicin was examined as shown in fig. 9, which shows the effect of sucrose addition in the medium on the yield (peak area) of taxicin. The addition of sucrose to the medium greatly increased the yield of taxicin compared to the control medium.
Example 7
Experiment for optimizing addition amount of mixed carbon source
1. Selection of fermentation strains: burkholderia order [ Polyangium ] brachysporum DSM7029 contains epothilone gene cluster strain MMR 11.
2. Activation of the strain: the strain is inoculated on a solid plate culture medium, cultured for 2 days at 30 ℃, then inoculated in a liquid culture medium, and cultured for 2 days at 30 ℃ and 200 rpm. The cells were then transferred to a small triangular flask and cultured at 30 ℃ and 200rpm for 2 days. Then, the bacterial liquid is collected for later use.
Wherein,
the solid plate culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. The solid medium was added with 1.5% agar and heat-sterilized at 121 ℃ for 20 minutes.
The liquid culture medium comprises the following components: casein peptone 8g/L, yeast powder 4g/L, magnesium chloride hexahydrate 4.06g/L, glycerol 5mL, pH 7. Moist heat sterilization at 121 ℃ for 20 minutes.
3. A fermentation culture mode: and (3) inoculating 25mL of the bacterial liquid prepared in the step (2) into a fermentation medium, and culturing at 30 ℃ and 200rpm for 6 days.
Wherein,
the fermentation medium comprises the following components in percentage by weight: 8g/L of casein peptone, 4g/L of yeast powder, 4.06g/L of magnesium chloride hexahydrate, 5mL of glycerol, 5g/L of sucrose, 0-10g/L of glucose, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 1mL/L of trace element solution and 20mL/L of amberlite XAD-16 macroporous adsorption resin.
In the trace element solution, the content of manganese chloride tetrahydrate is 7.9g/L, the content of zinc sulfate heptahydrate is 1.5g/L, the content of copper sulfate pentahydrate is 6.4g/L, and the content of ferrous sulfate heptahydrate is 1.1 g/L.
4. Separation, extraction, purification and detection of epothilone: after 6 days of fermentation, the resin was collected by filtration through a 100 mesh screen and extracted twice overnight in a triangular flask with the addition of 25mL of methanol. After being combined, 2mL of leaching liquor is taken, filtered by a 0.22 mu m filter and detected by mass spectrum.
5. And (4) calculating the yield of the natural product according to a curve of the relation between the absorption peak area and the concentration of the standard sample, and averaging the three groups of results and calculating the error.
The production of epothilones was examined as shown in FIG. 10. The results showed that addition of 5g/L glucose based on the fermentation medium containing 5g/L sucrose produced 2.87 times the yield without glucose. The yield of 10g/L glucose added to the fermentation medium containing 5g/L sucrose was 3.45 times that of the medium without glucose. Compared with a single carbon source, the mixed carbon source can obviously improve the yield of the epothilone.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (27)
1. A culture medium for preparing natural products by fermentation of a Burkholderia DSM7029 strain, which comprises casein peptone, yeast powder, magnesium chloride hexahydrate, glycerol, sodium acetate, sodium propionate, methylmalonic acid, a trace element solution and a macroporous adsorption resin, and also comprises an additional carbon source component which is selected from one or more of sucrose, starch, dextrin, glucose and glycerol.
2. The culture medium of claim 1, wherein the composition of the solution of trace elements comprises at least manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, and ferrous sulfate heptahydrate.
3. The culture medium according to claim 1, wherein the macroporous adsorbent resin is amberlite XAD-16 macroporous adsorbent resin.
4. The culture medium of claim 1, wherein the sucrose is added in an amount of 5-50g/L in the additional carbon source component.
5. The culture medium of claim 1, wherein the starch is added in an amount of 5-50g/L in the additional carbon source component.
6. The culture medium of claim 1, wherein the dextrin is added in an amount of 5-50g/L in the additional carbon source component.
7. The culture medium of claim 1, wherein the glycerol is added in an amount of 5-50mL/L in the additional carbon source component.
8. The culture medium according to claim 1, wherein the sucrose and the glucose are added in an amount of 5 to 50g/L in the additional carbon source component.
9. The culture medium of claim 1, wherein the sucrose is added in an amount of 25g/L in the additional carbon source component.
10. The culture medium of claim 1, wherein the starch is added in an amount of 25g/L in the additional carbon source component.
11. The culture medium of claim 1, wherein the dextrin is added in an amount of 25g/L in the additional carbon source component.
12. The culture medium of claim 1, wherein the glycerol is added in an amount of 25mL/L in the additional carbon source component.
13. The culture medium according to claim 1, wherein the sucrose is added in an amount of 5g/L and the glucose is added in an amount of 10g/L in the additional carbon source component.
14. The culture medium according to claim 1, wherein the content of the casein peptone in the culture medium is 8g/L, the content of the yeast powder is 4g/L, the content of the magnesium chloride hexahydrate is 4.06g/L, the content of the glycerol which is not additionally added is 5mL/L, the content of the sodium acetate is 50mg/L, the content of the sodium propionate is 100mg/L, the content of the methylmalonic acid is 100mg/L, the content of the trace element solution is 1mL/L, and the content of the macroporous adsorption resin is 20 mL/L.
15. The culture medium of claim 2, wherein the solution of trace elements contains 7.9g/L of manganese chloride tetrahydrate, 1.5g/L of zinc sulfate heptahydrate, 6.4g/L of copper sulfate pentahydrate, and 1.1g/L of ferrous sulfate heptahydrate.
16. A culture medium for improving autolysis of a Burkholderia DSM7029 strain in a thallus fermentation process is characterized by comprising casein peptone, yeast powder, magnesium chloride hexahydrate, glycerol, sodium acetate, sodium propionate, methylmalonic acid, a trace element solution and macroporous adsorption resin, and also comprising cane sugar which is additionally added.
17. The culture medium of claim 16, wherein the composition of the solution of trace elements comprises at least manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, and ferrous sulfate heptahydrate.
18. The culture medium according to claim 16, wherein the macroporous adsorbent resin is amberlite XAD-16 macroporous adsorbent resin.
19. The culture medium according to claim 16, wherein the sucrose is added in an amount of 5 to 50 g/L.
20. The culture medium according to claim 16, wherein the sucrose is added in an amount of 25 g/L.
21. The culture medium according to claim 16, wherein the content of the casein peptone is 8g/L, the content of the yeast powder is 4g/L, the content of the magnesium chloride hexahydrate is 4.06g/L, the content of the glycerol which is not additionally added is 5mL/L, the content of the sodium acetate is 50mg/L, the content of the sodium propionate is 100mg/L, the content of the methylmalonic acid is 100mg/L, the content of the trace element solution is 1mL/L, and the content of the macroporous adsorption resin is 20 mL/L.
22. The medium according to claim 15, wherein the solution of trace elements contains 7.9g/L of manganese chloride tetrahydrate, 1.5g/L of zinc sulfate heptahydrate, 6.4g/L of copper sulfate pentahydrate, and 1.1g/L of ferrous sulfate heptahydrate.
23. Use of a medium according to any one of claims 1-22 for the fermentative preparation of a natural product, wherein the burkholderia DSM7029 strain is polycystic brevibacterium [ Polyangium ] brachyspora DSM 7029.
24. Use according to claim 23, characterized in that said natural products are products of gene clusters constituted by polyketide synthases and/or non-ribosomal peptide synthetases and derivatives thereof.
25. The use of claim 23, wherein said natural products comprise at least an epothilone and a taxicin.
26. A method for increasing the yield of a natural product produced by fermentation of a strain of burkholderia DSM7029, characterized in that the method employs a medium according to any of claims 1-15 as the fermentation medium for the strain of burkholderia DSM 7029.
27. A method for improving autolysis of the order burkholderia DSM7029 strain during fermentation of the bacterial mass, wherein the method employs the medium according to any of claims 16-22 as the fermentation medium for the order burkholderia DSM7029 strain.
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| US4916063A (en) * | 1986-09-18 | 1990-04-10 | Bristol-Myers Company | BU-2867T peptide antibiotics |
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