CN106916810A - One kind rapidly and efficiently screens farnoquinone（MK‑7）The method of Producing Strain - Google Patents
One kind rapidly and efficiently screens farnoquinone（MK‑7）The method of Producing Strain Download PDFInfo
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- CN106916810A CN106916810A CN201710309437.7A CN201710309437A CN106916810A CN 106916810 A CN106916810 A CN 106916810A CN 201710309437 A CN201710309437 A CN 201710309437A CN 106916810 A CN106916810 A CN 106916810A
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- farnoquinone
- mutagenesis
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- respiratory chain
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- PFRQBZFETXBLTP-RCIYGOBDSA-N 2-[(2e,6e,10e,14e,18e)-3,7,11,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaen-1-yl]-3-methyl-1,4-dihydronaphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 PFRQBZFETXBLTP-RCIYGOBDSA-N 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 32
- PFRQBZFETXBLTP-UHFFFAOYSA-N Vitamin K2 Natural products C1=CC=C2C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C(=O)C2=C1 PFRQBZFETXBLTP-UHFFFAOYSA-N 0.000 title abstract description 19
- 235000009464 menaquinone-7 Nutrition 0.000 title abstract description 19
- 239000011700 menaquinone-7 Substances 0.000 title abstract description 19
- RAKQPZMEYJZGPI-LJWNYQGCSA-N menaquinone-7 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 RAKQPZMEYJZGPI-LJWNYQGCSA-N 0.000 title abstract description 19
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 39
- 238000002703 mutagenesis Methods 0.000 claims abstract description 39
- 230000035806 respiratory chain Effects 0.000 claims abstract description 24
- 239000003112 inhibitor Substances 0.000 claims abstract description 22
- 235000013557 nattō Nutrition 0.000 claims abstract description 16
- 238000012216 screening Methods 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims description 40
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 244000063299 Bacillus subtilis Species 0.000 claims description 19
- 239000011521 glass Substances 0.000 claims description 14
- 235000011187 glycerol Nutrition 0.000 claims description 11
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 239000000725 suspension Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 8
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 8
- 229940066779 peptones Drugs 0.000 claims description 8
- 229940080817 rotenone Drugs 0.000 claims description 8
- JUVIOZPCNVVQFO-UHFFFAOYSA-N rotenone Natural products O1C2=C3CC(C(C)=C)OC3=CC=C2C(=O)C2C1COC1=C2C=C(OC)C(OC)=C1 JUVIOZPCNVVQFO-UHFFFAOYSA-N 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000002504 physiological saline solution Substances 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- SJJCQDRGABAVBB-UHFFFAOYSA-N 1-hydroxy-2-naphthoic acid Chemical class C1=CC=CC2=C(O)C(C(=O)O)=CC=C21 SJJCQDRGABAVBB-UHFFFAOYSA-N 0.000 claims description 5
- 235000015278 beef Nutrition 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 229940005561 1,4-benzoquinone Drugs 0.000 claims description 4
- 150000004782 1-naphthols Chemical class 0.000 claims description 4
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 2
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- 241000219094 Vitaceae Species 0.000 claims 1
- 235000021021 grapes Nutrition 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
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- 231100000219 mutagenic Toxicity 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 230000035772 mutation Effects 0.000 description 11
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 7
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- 102000004190 Enzymes Human genes 0.000 description 4
- 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 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000003097 anti-respiratory effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
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- 235000013305 food Nutrition 0.000 description 3
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- 229920001817 Agar Polymers 0.000 description 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical group CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 2
- 206010028400 Mutagenic effect Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
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- 235000013402 health food Nutrition 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011676 menaquinone-4 Substances 0.000 description 2
- 231100000243 mutagenic effect Toxicity 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000019143 vitamin K2 Nutrition 0.000 description 2
- 239000011728 vitamin K2 Substances 0.000 description 2
- HYPYXGZDOYTYDR-HAJWAVTHSA-N 2-methyl-3-[(2e,6e,10e,14e)-3,7,11,15,19-pentamethylicosa-2,6,10,14,18-pentaenyl]naphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 HYPYXGZDOYTYDR-HAJWAVTHSA-N 0.000 description 1
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 108010067028 Mitochondrial Permeability Transition Pore Proteins 0.000 description 1
- BKRPFTYHTOCYHT-UHFFFAOYSA-N N#[C-].CC#N Chemical compound N#[C-].CC#N BKRPFTYHTOCYHT-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003262 anti-osteoporosis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005468 ion implantation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 150000003721 vitamin K derivatives Chemical class 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
- 229940041603 vitamin k 3 Drugs 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
Farnoquinone is rapidly and efficiently screened the invention discloses one kind（MK‑7）The method of Producing Strain.Original strain is used into atmospheric pressure at room plasma（ARTP）Mutagenesis, and with farnoquinone analogue and respiratory chain inhibitor flat board seed selection mutagenic strain.By after 96 well culture plates fermentation secondary screening, superior strain farnoquinone content improves 200%.A collection of fast growth is selected, farnoquinone yield is high, inheritance stability, the bafillus natto strong to environment resistance.
Description
Technical field
The present invention relates to a kind of method for rapidly and efficiently screening farnoquinone (MK-7) Producing Strain, belong to biotechnology neck
Domain.
Background technology
Farnoquinone is the fat-soluble blood coagulation biostearin of a class, is aphthoquinone series compound, according to its molecular structure
The length difference of upper C-3 isoprene side chains has 14 kinds of forms, represents that (n refers to isoprene unit on side chain with MK-n
Number), MK-4 (MK-4) and menaquinone-7 (MK-7) are most commonly seen, and MK-7 is due to can quickly by intestinal absorption, in blood
The advantages of long half time in liquid, is widely studied.Farnoquinone has significant in terms of the prevention and treatment of osteoporosis
Effect, is referred to as " forth generation anti-osteoporosis product ".In addition, farnoquinone can also improve rapidly due to vitamin K
The bleeding that shortage causes, anti-arteriosteogenesis and osteoarthritis, antitumor, anti-aging repair damaging cells, treat parkinsonism
Deng being widely used in the fields such as medicine, food.The application of farnoquinone has obtained the accreditation of authoritative institution.Day in 2005
This approval farnoquinone is listed as osteosporosis resistant medicament.State Food and Drug Administration of China also arranges farnoquinone
Enter (state's food medicine prison note in " vitamin, the mineral cpd list " of " regulation (tentative) is declared and evaluated to nutritious supplementary pharmaceutical "
[2005] No. 202), can be used as the raw material of health food or nutrition fortifier.In January, 2008, farnoquinone passes through U.S.'s food
Drug Administration (FDA) is assert safely.2009, European Parliament and EU Council were determined in its all member state,
Farnoquinone can be used as the raw material (2009/345/EC) of health food or nutrition fortifier.
At present, the patent of domestic and international farnoquinone relates generally to three aspect contents, is on the one hand the excellent of fermentation process regulation and control
Change (EP1803820B1；CN104357355A)；On the one hand it is separation and Extraction (the CN103898175A of farnoquinone product；
US20060057688A1)；On the other hand it is bacterial screening, Groupe Danone Rierwei Co., Ltd (France) uses bacitracin or peroxide
Make lactic acid bacteria nature mutation (US7981657B2)；Shanghai Harmony Feed Co., Ltd. uses ultraviolet and nitrosoguanidine complex mutation,
Farnoquinone superior strain (CN102827798A) is screened with reference to Protoplast Fusion Technique.
At present, industrialized production farnoquinone is mostly all to be produced with chemical synthesis, due to Production by Microorganism Fermentation
Its yield is too low, high cost, it is impossible to improve yield, it is necessary to carry out mutagenesis screening as the main path of batch production.Bacterial strain
The method of screening is generally chemical mutagenesis, physical mutagenesis method, ion implantation mutagenesis method and Protoplast Mutation method etc..Wherein,
Atmospheric pressure at room plasma (ARTP) mutagenesis not only can carry out quick mutagenesis to microorganisms such as microalgae, bacterium, fungi, yeast,
And because its positive mutation rate is high, compared with classic mutagenesis mode, it is easier to obtain the good excellent mutant bacteria of genetic stability.
In most of gram-positive bacteriums, farnoquinone (menaquinone) is that intracellular unique endogenous lipophilic is anti-oxidant
Agent, is also indispensable electron transit mediator in respiratory chain.Its action effect is similar with CoQ, by maintaining mitochondrial DNA and base
The stabilization of cause promotes the existence of cell, and independent Anti-G value is played by adjusting cell mitochondrial Permeability Transition Pore etc..
The present invention intends carrying out the starting strain of bafillus natto mutagenesis using ARTP methods, and for anti feedback mutant strain (knot
Structure analog resistant mutation bacterium) and the screening of bacterial strain of anti-respiratory chain inhibitor carried out a series of experiments, by contrasting substrate
The aspects such as wear rate, Biomass and yield, so that superior strain is obtained, for the industry that the microorganism for realizing farnoquinone is developed
Change lays the foundation.
The content of the invention
The technical problem to be solved in the invention is that strain improvement process efficiency is too low in the prior art, it is impossible to obtain
Superior strain；In order to solve the problem, the present invention provides a kind of method for rapidly and efficiently screening farnoquinone (MK-7) Producing Strain.
Specifically, this programme is：
A kind of method for rapidly and efficiently screening farnoquinone Producing Strain, wherein, with bafillus natto as starting strain, warp
Atmospheric pressure at room plasma (ARTP) mutagenesis original strain, the bacterial strain after mutagenesis is selected with the flat board containing farnoquinone analogue
After educating, then seed selection is carried out on the flat board containing respiratory chain inhibitor, and through 96 well culture plates fermentation secondary screening, obtain farnoquinone high
Produce bacterium.
Method of the present invention, wherein, concretely comprise the following steps：
(1) bafillus natto starting strain is activated in seed culture medium；
(2) the metal slide glass of sterilizing is placed in sterilized glass plate, the Bacillus natto bacteria suspension after activation is uniform
Coat on slide glass, carry out mutagenesis in ARTP-II type mutagenesis machines respectively；
(3) after the completion of the mutagenesis of Bacillus natto bacteria suspension, it is eluted with physiological saline, and is coated on addition farnoquinone structure
In analog aseptic flat board, pick out single bacterium colony and fermented in 96 well culture plates, farnoquinone superior strain is carried out into preservation；
(4) after the bafillus natto activation for obtaining step (3), it is uniformly coated on addition respiratory chain inhibitor aseptic flat
On plate, pick out single bacterium colony and fermented in 96 well culture plates, farnoquinone superior strain is carried out into preservation.
Method of the present invention, wherein, in the step (2), the mutagenesis of atmospheric pressure at room plasma uses ARTP-II types
Mutagenesis machine, with the time as variable element, power setting is 100W, and throughput is set as 10SLM, adjustment mutation time is 30~
150s。
Method of the present invention, wherein, with the addition of 2.5~20mg/L vitamins in K2 analogue aseptic flat boards
K2 analogues, the similar thing of the farnoquinone is 1- hydroxy-2-naphthoic acids, 2-MNQ, 1,4-benzoquinone, 1- naphthalenes
One or more in phenol.
Method of the present invention, wherein, in the step (4), 5 are with the addition of in addition respiratory chain inhibitor aseptic flat board
~200mg/L respiratory chain inhibitors, the respiratory chain inhibitor is one or more in rotenone and cyanide.
Method of the present invention, wherein, in the step (1), seed culture medium is 30g/L glucose, 40g/L albumen
Peptone, 5g/LNaCl, 5g/L beef extract, 5g/L yeast extracts, 37 DEG C of cultivation temperature, 120rpm cultures 1d.
Heretofore described farnoquinone is MK-7 types.
In a preferred embodiment, comprise the following steps：
(1) bafillus natto original strain is activated on seed culture medium；
(2) extension rate is calculated according to bacteria concentration, the OD of bacterium solution will be activated using the physiological saline after sterilizing600It is diluted to
Between 0.5~2.0, take 10ul bacterium solutions and be uniformly applied on metal slide glass, without air-drying, the metal slide glass that will be equipped with sample is moved to
ARTP mutagenesis systems operation storehouse carries out Mutagenesis experiments.Wherein, ARTP mutation times be 30s, 45s, 60s, 90s, 120s and 150s,
It is preferred that 60s.
(3) after the completion of the mutagenesis of Bacillus natto bacteria suspension, it is eluted with physiological saline, and is coated on addition farnoquinone structure
In analog aseptic flat board；
New bacteria suspension is carried out suitably to dilute, the primary dcreening operation flat board containing farnoquinone analogue is coated with, culture is counted,
Selecting eugonic single bacterium colony carries out 96 well culture plate fermentations, and preservation is carried out to farnoquinone superior strain；
Wherein, described farnoquinone analogue be 1- hydroxy-2-naphthoic acids, 2-MNQ, to benzene
Any one or a few mixture in quinone, 1- naphthols.Addition concentration is 2.5~20mg/L, preferably 5mg/L.
(4) after cultivating the farnoquinone superior strain that step 3 is obtained, appropriate dilution, coating suppress containing respiratory chain
The primary dcreening operation flat board of agent, culture is counted, and selecting eugonic single bacterium colony carries out 96 well culture plate fermentations, to farnoquinone Producing Strain
Strain carries out preservation；
Wherein, described respiratory chain inhibitor is any one or a few the mixing in rotenone and cyanide acetonitrile
Thing.The addition concentration of rotenone is 20~200mg/L, preferably 150mg/L；The addition concentration of acetonitrile is that 5~80mg/L is preferred
20mg/L。
(5) above-mentioned superior strain is carried out into Shaking culture.It is inoculated into equipped with 100mL fermentation mediums by 5% inoculum concentration
In 500mL triangular flasks with baffle plate, fermentation medium be 50g/L glycerine, 30g/L soy peptones, 0.6g/L dusty yeasts,
0.3g/LK2HPO4、0.1g/LCaCl2·H2O、0.3g/LMgSO4.7H2O, the 200rpm cultures 3d at 37 DEG C.
The beneficial effects of the present invention are：
(1) present invention takes the mutagenesis of atmospheric pressure at room plasma to make the structure of microorganism wall or cell membrane and lead to
Permeability changes, and causes gene damage, and the high activity energy particle that ARTP is rich in causes to damage to the inhereditary material of thalline, and
Induce cell and start SOS repair mechanisms.And abundant gene mutation site can be produced in repair process, and finally stable heredity is entered
And form mutant strain.It is high with positive mutation rate compared with classic mutagenesis mode, brief introduction is operated, mutagenesis low cost is applied widely
The advantages of with easily genetic stability good mutant bacteria is obtained.
(2) mutagenesis means disclosed in this invention carry out resistance screening in conjunction with analogue, can hinder metabolin
Normal generation, changed in orientation of the structure (feedback-inhibition resistance) of the enzyme of feedback inhibition, or the life for changing the enzyme for checking
Into system (anti feedback is checked), so only to analogue and insensitive mutant bacteria could continued growth, carry significantly
Screening frequency high.
(3) mutagenesis means disclosed in this invention carry out resistance screening in conjunction with respiratory chain inhibitor, can make natto bud
Spore bacillus respiratory chain is obstructed, and induction strain is oriented and is mutated to the direction of anti-respiratory chain inhibitor.The bacterial strain profile separated is big
Cause is divided into three kinds, the translucent colony of respectively white fold shape, all-transparent water-drop-shaped and film parcel.All-transparent water-drop-shaped and film
The transparent bacterium colony of high viscosity of parcel, has wire drawing situation to occur, and the MK-7 contents of white fold bacterium colony are higher.
Brief description of the drawings
6 three kinds of typical case's mutation thalli morphology comparison diagrams of Fig. 1 embodiments.
Specific embodiment
Embodiment 1：This example demonstrates that biological material source information of the invention
Biomaterial in the present invention is bafillus natto, is isolated from the natto product of in the market purchase.
Embodiment 2：The process of the present embodiment explanation ARTP mutagenesis
By bafillus natto on solid plate activation culture, the thalline after activation culture suspends with physiological saline.With
Thalline is suitably diluted to OD by physiological saline600Metal slide glass is placed in alcolhol burner flame envelope by value between 0.5~2.0 in super-clean bench
Calcination 30s, is put into sterilized glass plate after cooling, takes 10ul bacteria suspensions and is uniformly applied to slide glass, without air-drying, is lured
Become.
Ultraviolet sterilization 30min is first opened in ARTP mutagenesis systems operation storehouse, then is put to ARTP mutagenesis system slide glass with aseptic nipper
System operation storehouse, regulation microscope carrier lower section knob, makes slide glass be at flow ports 2mm.Setting instrument power is 100W, throughput ginseng
Number is 10SLM, and with mutation time as variable element, mutation time is 30s, 45s, 60s, 90s, 120s, 150s.Sample treatment is complete
Finish, slide glass is put into the EP pipes equipped with 490ul physiological saline, shake 5min, bafillus natto is eluted in liquid, shape
The bacteria suspension of Cheng Xin.
Result shows that process 30s, 45s, 60s, 90s, 120s and 150s through plasma jet, its fatal rate is respectively
19.89%th, 37.63%, 58.18%, 90.91%, 98.18% and 100%.
Embodiment 3：This example demonstrates that the process that analogue concentration critical value determines
By the thalline normal saline dilution after activation culture to 10-1~10-7Between, take the coating of 100ul dilutions flat
Plate.Wherein, the formula of solid plate is：30g/L glucose, 40g/L peptones, 5g/LNaCl, 5g/L beef extract, 5g/L yeast
Cream, 20g/L agar, 2.5~20mg/L 1,4-dihydroxy-2-naphthsaisyuoic acids, 1,4-benzoquinone, 2-MNQ or 1- naphthols.
Analogue can be combined due to similar to metabolite structure with repressor and allosteric enzymes, make the catalysis of enzyme
Effect is irreversibly suppressed.Determine bafillus natto using variety classes farnoquinone analogue as prescreening
A series of sensibility critical value, seed selection resistant mutant strain, the analog of concentration is added on flat board.
The farnoquinone analogue resistance concentration of table 1 is tested
| Concentration (mg/L) | 2.5 | 5 | 7.5 | 10 | 15 | 20 |
| 1- hydroxy-2-naphthoic acids | + | + | + | ± | ± | - |
| 1,4-benzoquinone | + | + | + | ± | ± | - |
| 2- methyl-1,4-naphthaquinones | + | + | ± | ± | - | - |
| 1- naphthols | + | + | + | + | ± | ± |
Note：“+”Good；“±”Bad；"-" ND
Drawn by above-mentioned result of the test, when the concentration of 1- hydroxy-2-naphthoic acids is less than 10mg/L, B.natto growths are good
It is good；When its concentration is higher than 20mg/L, B.natto does not grow.When the concentration of 2- methyl-1,4-naphthaquinones and 1- naphthols is respectively lower than
During 7.5mg/L and 15mg/L, B.natto well-growns.
Embodiment 4：This example demonstrates that the process based on farnoquinone analogue bacterium
On this basis, ARTP mutagenesis is combined with analogue, respectively to the bacteria suspension for the treatment of 30s, 60s and 90s
It is coated on the pre-sifted flat board containing 5mg/L, 10mg/L or 20mg/L.The single bacterium colony that picking grows accesses equipped with 0.5~1mL 96
Well culture plate top fermentation.Wherein, fermentative medium formula is：50g/L glycerine, 30g/L soy peptones, 0.6g/L dusty yeasts,
0.3g/LK2HPO4、0.1g/LCaCl2·H2O、0.3g/L MgSO4.7H2O.In 37 DEG C of shaking table, with the rotating speed of 200rpm,
Culture 72h, detects through high performance liquid chromatography.
HPLC methods：Chromatographic column：C18 posts, column temperature：50 DEG C, mobile phase：Methyl alcohol, flow velocity：1ml/min, ultraviolet detection wavelength
270nm, sample size：30ul, detection time：35min.
The Mutagenic Effect that the ARTP of table 2 is combined with analogue
In table result be it is each under the conditions of the highest MK-7 yield that filters out, by above-mentioned result of the test, most mutagenesis 60s at last
Thalline is coated on the flat board containing 5mg/L1- hydroxy-2-naphthoic acids as optimum condition.The mutant bacteria B.natto A1-1 of acquisition
MK-7 yield be 15.19mg/L, improve 83.90% than original bacteria.
Embodiment 5：This example demonstrates that the process that respiratory chain inhibitor concentration critical value determines
By the thalline normal saline dilution after activation culture to 10-1~10-7Between, take the coating of 100ul dilutions flat
Plate.Wherein, the formula of solid plate is：30g/L glucose, 40g/L peptones, 5g/LNaCl, 5g/L beef extract, 5g/L yeast
Cream, 20g/L agar, 20~200mg/L rotenone or 5~80mg/L acetonitriles.
Determine the sensibility critical value of bafillus natto, seed selection using variety classes respiratory chain inhibitor as prescreening
A series of resistant mutant strain, the analog of concentration is added on flat board.
The respiratory chain inhibitor resistance concentration of table 3 is tested
| Concentration (mg/L) | 20 | 50 | 80 | 100 | 150 | 200 |
| Rotenone | + | + | + | ± | ± | - |
| Concentration (mg/L) | 5 | 10 | 20 | 40 | 60 | 80 |
| Acetonitrile | + | + | ± | ± | - | - |
Note：“+”Good；“±”Bad；"-" ND.
By above-mentioned result of the test, when the concentration of rotenone is less than 100mg/L, B.natto well-growns；When its concentration is high
When 200mg/L, B.natto does not grow.When the concentration of acetonitrile is less than 20mg/L, B.natto well-growns；When its concentration is high
When 60mg/L, B.natto does not grow.Rotenone block electronics from transmission from NADH to CoQ, cyanide block electronics by cell
Transmission of the pigment aa3 to oxygen.And in thalline, the yield of farnoquinone is relevant with the development of strain and physiological status, depend on
Encode the activity height of the expression of related gene.Farnoquinone plays main electro transfer in the respiratory chain of B.natto and makees
With, if to improve the yield of farnoquinone, respiratory chain inhibitor is added in the medium, due to making respiratory chain be obstructed, so
Induction strain is oriented and is mutated to the direction of anti-respiratory chain inhibitor.
Embodiment 6：This example demonstrates that the process based on respiratory chain inhibitor bacterium
Will in embodiment 4 obtain bafillus natto it is activated after, dilution spread is containing 80mg/L, 150mg/L trifoliate jewelvine
On the pre-sifted flat board of ketone and 10mg/L, 20mg/L acetonitrile.The single bacterium colony that picking grows accesses 96 holes equipped with 0.5~1mL and cultivates
Plate top fermentation.Wherein, fermentative medium formula is：50g/L glycerine, 30g/L soy peptones, 0.6g/L dusty yeasts, 0.3g/
LK2HPO4、0.1g/LCaCl2·H2O、0.3g/LMgSO4.7H2O.In 37 DEG C of shaking table, with the rotating speed of 200rpm, culture
72h, detects through high performance liquid chromatography.
The Mutagenic Effect of the respiratory chain inhibitor of table 4
By above-mentioned result of the test, most the thalline of mutagenesis 60s is coated on the flat board containing 20mg/L acetonitriles as most suitable at last
Condition.The MK-7 yield of the mutant bacteria B.natto A5-1 of acquisition is 23.61mg/L, and 185.84% is improve than original bacteria；
The MK-7 yield of B.natto A13-12 is 25.51mg/L, and 208.84% is improve than original bacteria.Mutagenesis disclosed in this invention
The bacterial strain profile that means are separated is roughly divided into three kinds, and A, B, C three major types are designated as successively, respectively white fold shape A classes, complete
The translucent colony C classes of transparent water-drop-shaped B classes and film parcel, as shown in Figure 1.The high viscosity of all-transparent water-drop-shaped and film parcel
Transparent bacterium colony, has wire drawing situation to occur, and the MK-7 contents of white fold bacterium colony are higher.
Embodiment 7：Fermenting performance is investigated
The Producing Strain of -80 DEG C of preservations is seeded in the 500mL triangular flasks with baffle plate equipped with 100mL seed culture mediums,
At 37 DEG C, 120rpm shaken cultivations 1d.The seed liquor that will activate for two generations is inoculated into equipped with 100mL fermentation trainings by 5% inoculum concentration
Support in the 500mL triangular flasks with baffle plate of base, 200rpm is cultivated at 37 DEG C.Seed culture medium：Tryptone 10g/L, beef
Medicinal extract 5g/L, yeast extract 5g/L, glucose 30g/L, NaCl 5g/L, pH7.0,121 DEG C, 20min.Fermentation medium：50g/L
Glycerine, 30g/L soy peptones, 0.6g/L dusty yeasts, 0.3g/LK2HPO4、0.1g/LCaCl2·H2O、0.3g/
LMgSO4.7H2O, pH7.0,121 DEG C, 20min.
Result is as shown in table 6.
The different strains fermenting property comparison sheet of table 6
| Thalli morphology | Glycerine wear rate (g/L/h) | MK-7(mg/L) | ||
| Control (D) | White accordion | 0.89 | 30.65 | 8.26 |
| B.natto A1-1 | Transparent drops | 1.47 | 30.22 | 15.19 |
| B.natto A5-1 | White accordion | 1.00 | 38.21 | 23.61 |
| B.natto A13-12 | White accordion | 0.94 | 42.17 | 25.51 |
By above-mentioned result of the test, while carrying out fermenting property investigation to tri- plants of bacterium of B.nattoA1-1, A5-1 and A13-12.
Compared with original bacteria, the glycerine wear rate of mutant bacteria A1-1 reaches 1.47g/L/h in 24h, but its biomass is relatively low, maximum
OD600It is that 30.22, MK-7 yield is 15.19mg/L.Mutant bacteria A1-1 is transparent drops, and zymotic fluid color is relatively deep and more glues
Thick, bacterium solution has wire drawing, and the glycerine of its consumption is mostly used for the synthesis of other materials such as gamma-polyglutamic acid, cause MK-7 yield compared with
It is low.Mutant bacteria A5-1MK-7 yield is 23.61mg/L, and glycerine wear rate is similar to mutant bacteria A13-12, slow compared with A1-1,
During 24h, its glycerine wear rate is only 1.00g/L/h, and follow-up its glycerine wear rate is accelerated, and is consumed substantially during 72h completely.
A13-12 MK-7 yield highests, are 25.51mg/L.A13-12 biomass is larger, maximum OD600Reach 42.17.
Claims (6)
1. a kind of method for rapidly and efficiently screening farnoquinone Producing Strain, it is characterised in that with bafillus natto be the bacterium that sets out
Strain, through atmospheric chamber isothermal plasma（ARTP）Mutagenesis original strain, the bacterial strain after mutagenesis is used containing farnoquinone analogue
After flat board seed selection, then seed selection is carried out on the flat board containing respiratory chain inhibitor, and through 96 well culture plates fermentation secondary screening, obtain dimension life
Plain K2 Producing Strains.
2. method according to claim 1, it is characterised in that methods described is concretely comprised the following steps：
（1）Bafillus natto starting strain is activated in seed culture medium；
（2）The metal slide glass of sterilizing is placed in sterilized glass plate, by the Bacillus natto bacteria suspension even spread after activation
In on slide glass, mutagenesis is carried out in ARTP-II type mutagenesis machines respectively；
（3）After the completion of bafillus natto bacteria suspension mutagenesis, it is eluted with physiological saline, and be coated on addition farnoquinone knot
In structure analog aseptic flat board, pick out single bacterium colony and fermented in 96 well culture plates, farnoquinone superior strain is carried out into preservation；
（4）By step（3）After the superior strain activation of acquisition, it is uniformly coated in addition respiratory chain inhibitor aseptic flat board, chooses
Select single bacterium colony to be fermented in 96 well culture plates, farnoquinone superior strain is carried out into preservation.
3. method according to claim 1, it is characterised in that the step（2）In, atmospheric pressure at room plasma mutagenesis is adopted
With ARTP-II type mutagenesis machines, with the time as variable element, power setting is 100W, and throughput is set as 10SLM, adjusts mutagenesis
Time is 30 ~ 150s.
4. method according to claim 1, it is characterised in that with the addition of 2.5 ~ 20 in K2 analogue aseptic flat boards
Mg/L farnoquinone analogues, the similar thing of the farnoquinone be 1- hydroxy-2-naphthoic acids, 2-MNQ,
One or more in 1,4-benzoquinone, 1- naphthols.
5. method according to claim 1, it is characterised in that the step（4）In, addition respiratory chain inhibitor is aseptic flat
5 ~ 200 mg/L respiratory chain inhibitors are with the addition of in plate, the respiratory chain inhibitor is the one kind or many in rotenone and cyanide
Kind.
6. method according to claim 2, it is characterised in that the step（1）In, seed culture medium is 30 g/L grapes
Sugar, 40g/L peptones, 5g/L NaCl, 5g/L beef extracts, 5g/L yeast extracts, 37 DEG C of cultivation temperature, 120rpm cultures 1d.Fermentation
Culture medium prescription is 50g/L glycerine, 30g/L soy peptones, 0.6g/L dusty yeasts, 0.3g/LK2HPO4、0.1 g/
LCaCl2·H2O 、0.3g/LMgSO4.7H2O, 37 DEG C of cultivation temperature, incubation time 3d.
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| CN102827798A (en) * | 2012-09-14 | 2012-12-19 | 上海红马饲料有限公司 | Vitamin K2 high-yielding strain |
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| CN113755404A (en) * | 2021-10-13 | 2021-12-07 | 华北制药股份有限公司 | High-yield strain of vitamin K2(MK-7), screening method thereof and method for producing vitamin K2(MK-7) |
| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
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| CN114854732A (en) * | 2022-05-24 | 2022-08-05 | 南京工业大学 | Method for improving yield of vitamin K2 by regulating and controlling electron transfer chain |
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