CN105969702A - Serratia marcescens RZ 21-C6 and application thereof - Google Patents
Serratia marcescens RZ 21-C6 and application thereof Download PDFInfo
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- CN105969702A CN105969702A CN201610593964.0A CN201610593964A CN105969702A CN 105969702 A CN105969702 A CN 105969702A CN 201610593964 A CN201610593964 A CN 201610593964A CN 105969702 A CN105969702 A CN 105969702A
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- prodigiosin
- serratia marcescens
- xylose
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- 241000607715 Serratia marcescens Species 0.000 title claims abstract description 35
- HCOLPNRPCMFHOH-UHFFFAOYSA-N Prodigiosin Natural products CCCCCC1C=C(C=C/2N=C(C=C2OC)c3ccc[nH]3)N=C1C HCOLPNRPCMFHOH-UHFFFAOYSA-N 0.000 claims abstract description 63
- TWFGRJUTAULJPZ-USZBIXTISA-N prodigiosin Chemical compound N1=C(C)C(CCCCC)=C\C1=C/C1=NC(C=2[N]C=CC=2)=C[C]1OC TWFGRJUTAULJPZ-USZBIXTISA-N 0.000 claims abstract description 63
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 37
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 23
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000010902 straw Substances 0.000 claims abstract description 15
- 235000000346 sugar Nutrition 0.000 claims abstract description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 14
- 150000008163 sugars Chemical class 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 239000006052 feed supplement Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229910052603 melanterite Inorganic materials 0.000 claims description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000012807 shake-flask culturing Methods 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 239000012531 culture fluid Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 238000011218 seed culture Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 239000000758 substrate Substances 0.000 abstract description 12
- 238000003786 synthesis reaction Methods 0.000 abstract description 12
- 239000002028 Biomass Substances 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 3
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- 238000004321 preservation Methods 0.000 abstract description 3
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- 239000003814 drug Substances 0.000 abstract description 2
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- 241000894006 Bacteria Species 0.000 description 25
- 230000001580 bacterial effect Effects 0.000 description 22
- 230000035772 mutation Effects 0.000 description 18
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- 229960003487 xylose Drugs 0.000 description 14
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- 230000003505 mutagenic effect Effects 0.000 description 7
- 240000008042 Zea mays Species 0.000 description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 6
- 235000005822 corn Nutrition 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 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
- 241001052560 Thallis Species 0.000 description 4
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
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- 239000000194 fatty acid Substances 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000607720 Serratia Species 0.000 description 3
- 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 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 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 description 3
- 210000002808 connective tissue Anatomy 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
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- 150000003233 pyrroles Chemical class 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000004252 FT/ICR mass spectrometry Methods 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
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- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 230000000243 photosynthetic effect Effects 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- FYXXJXKLTCPPHW-UHFFFAOYSA-N 2-methoxy-1h-pyrrole Chemical group COC1=CC=CN1 FYXXJXKLTCPPHW-UHFFFAOYSA-N 0.000 description 1
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000842 anti-protozoal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
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- 230000005494 condensation Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
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- 238000010168 coupling process Methods 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
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- 238000009630 liquid culture Methods 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- -1 phospholipid Fatty acid Chemical class 0.000 description 1
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- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
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- 239000013589 supplement Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Classifications
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/425—Serratia
- C12R2001/43—Serratia marcescens
-
- 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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses serratia marcescens RZ 21-C6 and application thereof. The preservation number of the serratia marcescens RZ 21-C6 is CGMCCNo.12714. The mutant strain has the advantages that the utilization efficiency of a substrate, the genetic stability and the yield of prodigiosin are high, and the utilization effect of the mutant strain on xylose is particularly good. In a 5L fermentation tank, pesticide residue straw hydrolysate with xylose as a main component is low in price, a biomass carbon source is developed for production of the prodigiosin, the yield of the prodigiosin can reach 15-20 g/L during multi-batch fermentation, and the conversion rate of total reducing sugar directly used for prodigiosin synthesis is 20% or above. The method for producing the prodigiosin through the serratia marcescens at normal pressure and room temperature by means of plasma mutant strain and the utilization of the xylose and the pesticide residue straw hydrolysate by the serratia marcescens can promote achievement of prodigiosin industrialization, and the method can be used for the fields of biological medicines, ecological environmental management, agricultural biological controlling and the like.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the serratia marcescens after a strain atmospheric pressure at room plasma mutation and efficiently utilize
Xylose or the method for agriculture residual straw hydrolyzed solution high yield prodigiosin.
Background technology
Prodigiosin is the general name of a natural red colouring matter family, the methoxypyrrole skeleton that its basic structure is made up of three pyrrole rings,
Molecular formula is C20H25N3O, molecular weight is 323.2Da.Find also when studying Serratieae growth from nineteen twenty-nine Amako etc.
By Harashima etc. since nineteen sixty separates acquisition first, this physical property and bioactive research are received much concern always.
In addition to as a kind of natural biological dye, its many biologic activity are also recognized by gradually people, including antibacterium,
Malaria, antifungal and antiprotozoal activity.A large amount of reports also show that it is in the water pollution control of body eutrophication and agriculture
Also using value is had in industry biological and ecological methods to prevent plant disease, pests, and erosion.Along with finding apoptotic research in recent years, prodigiosin can cause the apoptosis of cancerous cell
And the least to Normocellular toxicity, it is a kind of good cancer therapy drug.National cancer institute finds the denseest especially
The prodigiosin of degree has obvious resistance effect to 57 kinds of different tumor cells of people.
Prodigiosin is as a kind of secondary metabolites, and it produces mainly by Microbe synthesis.Nature produces the bacterium of prodigiosin
Strain mainly includes some special marine bacteria etc. of serratia marcescens, actinomycetes, pseudomonas, and discovered in recent years.
Wherein with the most extensive to the research of serratia marcescens.Prodigiosin product is to pass through in the biosynthesis of serratia marcescens
Two bifidum pathway complete, and precursor is a single pyrroles precursor 2-methyl-3-amyl group pyrroles and double pyrroles's precursor 4-methoxy
Base-2,2-bis-pyrroles's-5-carboxaldehyde, form linear tripyrrole compound prodigiosin through condensing enzyme condensation coupling.Spirit bacterium is red
The synthesis of element is completed by pig gene cluster, and this gene cluster includes 16 genes, and wherein to be directly responsible for spirit bacterium red for 14 genes
Element and the synthesis of precursor thereof, two other gene is responsible for regulating and controlling this biosynthetic process.This shows that the synthesis of prodigiosin is one
Individual controlled by multiple genes, by the complex biological process of multifactor impact.
The yield of Production by Microorganism Fermentation prodigiosin varies at present, and in document report, yield is scarcely higher than 1g/L, the fewest
Number breaks through the yield of 10g/L.This is extremely complex mainly due to the condition impact of serratia marcescens synthesis prodigiosin.Cause
The biggest reason of prodigiosin volume variance essentially from the culture medium substrate component such as strain differences, carbon nitrogen source, temperature, pH value,
Ventilation etc..And the difference of the most different substrates and production bacterial strain itself is maximum to pigment composition and yield effect.Especially depend on
The utilization of low value carbon source and the aspect that efficiently synthesizes of prodigiosin are still had greatly by the serratia marcescens relying the characteristic in bacterial strain self
Room for promotion.It is worth mentioning that the relevant report that the most xylose is not done substrate synthesis prodigiosin at present, and xylose is main
Being the monomer of the lignocellulose biomass main component xylan the abundantest as large nature, exploitation can efficiently utilize with wood
Sugar has become as renewable chemicals for the industrial producing strain of the inheritance stability of the lignocellulose biomass hydrolyzed solution of main component
Study hotspot with energy organism refining field.
Study condition and technology be less demanding, simple and efficient, effect outstanding feature because having for the mutagenic breeding of microorganism, becomes experiment
One of room and enterprise's most common method.Serratia marcescens carries out mutagenic breeding also have been reported that with the yield improving prodigiosin,
As lithium chloride and ethyl methane sulfonate process, ultraviolet mutagenesis and microwave irradiation etc., but effect is limited.And atmospheric pressure at room plasma by
, active particle concentration low in own temperature is high, wide variety and act on microorganism and can change its permeability and cause gene simultaneously
Damage, thus it is expected to cause the change of microbial gene sequences and metabolism network thereof, cause microorganism to have on bigger probability
Benefit the direct mutation of production.
It is starting strain that the present invention utilizes a strain of this laboratory screening to produce prodigiosin serratia marcescens, and early stage is by after fermentation optimization
Prodigiosin yield is 2.64g/L, however it remains the defects such as yield is on the low side, bacterial strain is easily degenerated, and hereditary stability is poor.The present invention
Intend carrying out mutagenic breeding by atmospheric pressure at room plasma, it is desirable to solve above-mentioned bacterial strains hereditary stability poor, prodigiosin yield
The highest, under-utilized to cheap biomass carbon source problem, especially lacks a large amount of agricultures residual thing straw hydrolyzed solution containing xylose
Utilization ability.
Summary of the invention
It is an object of the invention to according to above-mentioned deficiency present in prior art, it is provided that a kind of serratia marcescens
CGMCCNo.12714, and use the method that this bacterial strain efficiently produces prodigiosin.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
One plant height produces the mutant strain of prodigiosin, entitled serratia marcescens (Serratia marcescens) RZ 21-C6, preservation
Numbered CGMCCNo.12714.The acquisition of described bacterial strain comprises the steps:
(1) by the fermentation liquid normal saline dilution of serratia marcescens original strain cultivated through shaking table to 106-107Individual/mL, takes
10 μ L are coated on ARTP mutation instrument metallic carrier, and parameter is set to: throughput 10-15L/min, power 80-120
W, the process time is 15-240s.After mutation terminates, with aseptic nipper sheet metal is put into equipped with 1mL normal saline centrifugal
Guan Zhong.Shake 1-2min on the oscillator, the microorganism being attached on metal slide glass is eluted formation bacteria suspension.
(2) obtained bacteria suspension is carried out suitable gradient dilution, take 0.1mL diluent and be applied to containing fermentation liquid culture medium
On flat board, 28-32 DEG C of incubator cultivates 24-48h, does fatality rate curve.
(3) choose fatality rate more than 95%, to process the redness of time be better than single colony inoculation of original strain to 96 holes containing xylose
On flat board, lucifuge is cultivated, and detects its Biomass and amount of pigment, filter out pigment production and Biomass is significantly higher than other after 24-48h
Bacterial strain.
(4) obtained optimum strain continuous fermentation cultivates 10 generations more than to identify its stability, it is thus achieved that stable enhanced variant be
Serratia marcescens CGMCCNo.12714.
Mutant strain of the present invention utilizes the method for xylose or agriculture residual straw hydrolyzed solution fermenting and producing prodigiosin to comprise the steps:
(1) in the primary-seed medium of inoculation 2-3 ring serratia marcescens CGMCCNo.12714 to 30mL, this cultivation basigamy
Side is yeast extract 5g/L, and peptone 10g/L, NaCl 10g/L, pH are 7.0,28-32 DEG C, shaking flask rotating speed 150-300rpm
Cultivate 12-24h to OD600=1.0.
(2) take above-mentioned primary seed solution, be seeded to 30mL secondary seed medium by the inoculum concentration of 3%-5%, this culture medium prescription
Within xylose 20g/L, peptone 25g/L, FeSO4·7H2O 0.23mM and CaCl216.68mM, utilize CaCO3
PH is in 6.0-8.5,3%-5% inoculum concentration, at 28-32 DEG C of condition 150-300rpm lucifuge shake-flask culture 24-48h in control；
(3) the secondary seed culture fluid of inoculation 3%-5% ferments in 5L fermentation tank and produces prodigiosin, liquid amount 3L, primary carbon source
Within xylose or agriculture residual straw hydrolyzed solution total reducing sugars concentration 20g/L, peptone 25g/L, FeSO4·7H2O 0.23mM
And CaCl216.68mM, when culture medium middle peasant residual straw hydrolyzed solution total reducing sugars initial concentration is consumed to below 5g/L, single
Directly supplementing and concentrating agriculture residual straw hydrolyzed solution is original initial concentration to total reducing sugars concentration, and feed supplement number of times does not limits, and fermentation temperature is
28-32 DEG C, utilizing HCl and NaOH to control pH is 300-400rpm at 6.0-8.5, dissolved oxygen 40-60%, rotating speed, and lucifuge is sent out
Ferment 150-200h.
Compared with prior art, there is advantages that
(1) by ARTP mutagenic breeding, it is thus achieved that a strain character is excellent, hereditary stability is high, the Serratia of high yield prodigiosin
Mutant strain.
(2) the serratia marcescens CGMCCNo.12714 that the present invention obtains can not only efficiently utilize the carbon sources such as traditional sucrose, and
And xylose and the blending ingredients fermenting and producing prodigiosin with other monosaccharide thereof can also be utilized.
(3) the residual straw biomass of cheap agriculture with corn stalk hydrolysis as representative is exploited for the production of prodigiosin first, through sending out
Ferment feeding strategy optimizes, and prodigiosin concentration is up to 15-20g/L.
Accompanying drawing explanation
Fig. 1 is the fatality rate curve of serratia marcescens ARTP mutation.
Fig. 2 is the comparison of 19 strain direct mutation bacterial strain thalli growths and pigment synthesis.
Fig. 3 is the ultraviolet-visible absorption spectroscopy of prodigiosin.
Fig. 4 is the high resolution mass spec figure (FT-ICR-MS) of prodigiosin.
Fig. 5 is thalli growth and the hereditary stability analysis of pigment synthesis of mutant bacteria.
Fig. 6 is the TEM microexamination of original strain and mutant strain.
Fig. 7 is the cell membrane permeability analysis (A figure is adventitia, and B figure is inner membrance) of original strain and mutant strain.
Fig. 8 is the conditional curve of simulation muscovado fermenting and producing prodigiosin.
Fig. 9 is corn stalk hydrolysis fermenting and producing prodigiosin (A figure is single batch, and B figure is multiple batches of).
Bacterial strain RZ 21-C6 in the present invention, is preserved in China culture presevation administration committee common micro-organisms center, and address is: Beijing
North Star West Road, Chaoyang District, city 1 institute 3, Institute of Microorganism, Academia Sinica；Deposit number is CGMCC No.12714,
Classification And Nomenclature: serratia marcescens Serratia marcescens；Preservation date is on June 28th, 2016.
Detailed description of the invention
The present invention is explained further below in conjunction with embodiment, but the present invention is not limited in any form by embodiment.
The ARTP mutation of embodiment 1 high yield prodigiosin bacterial strain and screening
(1) strain mutagenesis: the atmospheric pressure at room plasma mutation fatality rate curve of serratia marcescens original strain as shown in Figure 1 (should
Original strain is isolatable from commercially available Aquatic product photosynthetic Bacteria agent, numbering RZ 21, is specifically shown in document: Min Taoling, Yang Qiyin, former driving,
Deng. the separation of a strain serratia marcescens and qualification in photosynthetic bacteria microbial inoculum. Agriculture of Anhui science, 2006,34 (22): 5837-5838,
5841.), plasma is stronger to serratia marcescens lethality, processes 15s and can kill the thalline of nearly 60%；Process 60s
Fatality rate later reaches more than 90%；Process 240s fatality rate and reach 100%.Sudden change itself has randomness, its fatality rate and
Relation between direct mutation is not fully aware of, but fatality rate is the highest according to the literature, it is thus achieved that the probability of positive mutation rate will be got over
Greatly.This research, in order to obtain the bacterial strain of higher positive mutation rate and be easy to select single bacterium colony, chooses fatality rate condition more than 96%
Processing, the time that i.e. processes is respectively 120s, 150s and 180s tri-process, the fatality rate that the these three process time is corresponding
It is respectively 96.3%, 97.9% and 98.8%.
(2) bacterial strain screening: select 19 strain colors in above-mentioned three kinds of flat boards processed red in the direct mutation bacterial strain of original bacteria, wherein
Direct mutation bacterial strain 4 strain of 120s, the direct mutation bacterial strain of numbered A1-A4,150s has 6 strains, numbered B1-B6,180s
Direct mutation bacterial strain have 9 strains, numbered C1-C9.By to above bacterial strain after 48h cultivated by the 96 hole flat boards containing xylose
OD535And OD600Comparison find that the pigment production ability of C6 bacterial strain and thalli growth are all best (Fig. 2).
(3) mutant bacteria chromogenic element qualitative: the mutagenic strain chromogenic element of C6 is dissolved in acid chloroform (pH 3.0) at 200-800nm ripple
Long lower scanning, collection of illustrative plates is as shown in Figure 3.Its specificity absworption peak peak value is at 535nm, and in reporting with document, prodigiosin is in acid
Detection wavelength 535nm under the conditions of property is completely the same, there is not the miscellaneous peak of other material simultaneously, tentatively concludes the produced redness of C6
Element is prodigiosin.Further with FTICRMS high resolution mass spectrum, pigment purification of samples is carried out identification and analysis, and collection of illustrative plates shows
It exists size is 324.20730Da [M+H+] Materials debris (Fig. 4), this molecular weight of material Mr=[M+H+]-1=323.2073
With prodigiosin C20H25N3The molecular weight Mr=323.2 of O is completely the same, finally determines that in sample, pigment is prodigiosin.
(4) mutant strain hereditary stability is analyzed: in succeeding generations, the mutant to each generation carries out Biomass growth and prodigiosin yield
Analysis, carried out the shake flask fermentation of 24-48h from 1 generation to 10 generations, have detected relative thalli growth now and pigment production,
The serratia marcescens mutant two indices of plasma mutation 10 instead of in all keep good hereditary stability (Fig. 5).
Embodiment 2 mutant strain physiological property is analyzed
(1) tem observation of cellular morphology change: observe Serratia marcescans mutational strain RZ 21-C6, its shape under transmission electron microscope
State has significant change (Fig. 6) compared with starting strain RZ21.The thalline volume of mutant bacteria substantially becomes big, and cell specific surface area becomes
Greatly；Cell wall becomes the thinnest, and cell surface starts to become mellow and full penetrating, and permeability increases, and this intracellular that will be highly advantageous to is secondary
Prodigiosin is secreted into outside born of the same parents, reduces the injury effect to self, is simultaneously also beneficial to the isolated and purified process of later product.
(2) cell membrane phospholipid Analysis of Fatty Acids Composition: as can be seen from Table 1, in mutant strain RZ 21-C6 somatic cells membrane phospholipid
Fatty acid composition there occurs bigger change: wherein satisfied fatty acid C15:0, C16:0 and C17:0 content is all in being greatly decreased,
Reduction amplitude is respectively 34.22%, 18.91% and 21.16%.The content of unsaturated fatty acid 16:1,17:1 and C18:1 then divides
Do not add 84.20%, 19.62% and 53.85%.Unsaturation in ARTP mutation, serratia marcescens membrane phospholipid composition
Content of fatty acid dramatically increases, and saturated fatty acid content is then greatly decreased, and above change can ultimately result in the increasing of cell membrane fluidity
Adding, beneficially intracellular organic matter transports away and the outer substrate of born of the same parents is conveyed into, thus strengthens the substrate utilization efficiency of bacterial strain and reduce secondary
The level metabolite toxic action to thalline self.
(3) cell membrane permeability analysis: use two kinds of methods of NPN and ONPG to have studied Serratia before and after mutagenic treatment respectively
The change of the interior outer membrane permeability of bacterium cell.As shown in Figure 7 A, the fluorescent value of original strain adventitia is significantly lower than mutant strain
, the permeability that the light absorption value of similar result inner membrance the most in figure 7b shows mutant bacteria is above original bacteria.Prominent
Change bacterial strain is compared with original strain, and on interior adventitia, permeability is all notable rising.High membrane permeability is conducive to the outer substrate of born of the same parents
Renewal and the secretion of metabolite.Result has led to mutant strain can utilize substantial amounts of born of the same parents outer substrate synthesis prodigiosin.
Effective secretion of secondary metabolites then can reduce intracellular substrate and coerce cell, thus form that prodigiosin efficiently synthesizes excellent
Gesture circulates.
(4) physiological change brief summary: through ARTP mutation, serratia marcescens there occurs significant change on physiology and appearance.First it is
Change the specific surface area of its cell so that it is be more conducive to metabolite and the transport of substrate；Its secondary cell wall is the most thinning, cell membrane
Mobility strengthens, and interior outer membrane permeability increases, and according to the literature, generally accumulates around cell membrane after prodigiosin synthesis,
It is highly beneficial that these physiological changies of mutant bacteria get rid of cell for a large amount of products, is simultaneously also beneficial to the entrance that substrate is convenient
Cell.Therefore, the mutant strain making serratia marcescens is utilized substrate to synthesize higher concentration by above-mentioned physiological characteristics more efficiently
Prodigiosin.
Table 1 original strain and mutant strain cell membrane fat acid component analysis
Note: standard substance content calculates according to 100%.
The utilization power of different carbon source is compared by embodiment 3 mutant bacteria and original bacteria
(1) investigate mutagenic strain RZ 21-C6 and 4 kinds of basic carbon sources (sucrose, glucose, xylose and arabinose) are utilized feelings
Condition is as shown in table 2.Compared with original strain, mutant strain is the output increased of prodigiosin 2.59 in the culture medium containing sucrose
Times, production efficiency improves 2.63 times, shows that the bacterial strain filtered out not only pigment production uprises, and production efficiency also improves,
This conclusion drawn with the physiological change of embodiment 2 is consistent.In the culture medium containing xylose, RZ 21-C6 produces the amount of prodigiosin
Improve 17.21 times than RZ 21, reach 3.27g/L.Glucose production pigment can not be utilized, sudden change compared to original strain
Bacterial strain RZ 21-C6 can well utilize the chromogenic element of glucose, and amount of pigment reaches as high as 1.44g/L.Different from other sugars
Be that mutant bacteria utilizes the situation change of arabinose synthesis prodigiosin little, but be more beneficial for the growth of thalline, now give birth to
Thing amount adds 25.25%.
(2) fermentation results based on above-mentioned various carbon sources, simulate corn straw water with a certain proportion of mixing carbon source in 5L fermentation tank
Solve the fermentation (total sugar 20g/L, glucose, xylose and three kinds of monosaccharide of arabinose are respectively 1.58,17.54 and 0.88g/L) of liquid,
During 48h fermentation ends, prodigiosin Cmax is 5.29g/L, and production efficiency reaches 0.11g/L/h (Fig. 8).At sweat
In, mutant bacteria is very fast to the utilization of glucose and arabinose, is the most exhausted in 30h.Data above shows with Semen Maydis
Straw hydrolyzed solution is that the hydrolyzing biomass cellulose of representative do carbon source through fermentation to produce prodigiosin is feasible in theory.
Table 2 mutant bacteria and original bacteria be the comparison of fermenting and producing prodigiosin in different carbon source
Note: carbon source initial concentration is 20g/L, incubation time is that 64h, ND represent and be not detected by.
Embodiment 4 mutant strain utilizes corn stalk hydrolysis fermenting and producing prodigiosin
(1) in 5L fermentation tank, investigating the prodigiosin list Batch fermentation that initial total reducing sugars concentration is 20g/L, acquired results is such as
Shown in Fig. 9 A, after fermentation 48h, prodigiosin maximum concentration reaches 4.98g/L, and production efficiency is 0.10g/L/h, and thalline is
Big OD600Value is 1.53 (36h).It should be noted that from fermentation diagram it can be seen that mutant bacteria is the most permissible in 48h
Easily run out of all reducing sugar, and prodigiosin change of production trend still has the space of lifting further.In order to realize spirit bacterium
The high concentration of red pigment produces, it is necessary to investigate the effect that corn stalk hydrolysis is fermented by feeding strategy further.
(2) as shown in Figure 9 B, the present embodiment utilizes fed-batch mode fermenting and producing prodigiosin, in corn stalk hydrolysis
When reducing sugar is consumed to below 5g/L, single directly supplements concentrating hydrolysate to 20g/L, feed supplement 4 times, and total reducing sugars concentration is
80g/L, to last feed supplement reducing sugar exhaust shared time 132h, now prodigiosin maximum concentration reaches 16.17g/L, produce
Efficiency reaches 0.12g/L/h, and total sugar conversion ratio is 20.21% (w/w), and the high concentration that can realize prodigiosin produces.
Claims (4)
1. serratia marcescens (Serratia marcescens) RZ 21-C6, deposit number is CGMCCNo.12714.
2. the application in producing prodigiosin of the serratia marcescens CGMCCNo.12714 described in claim 1.
Application the most according to claim 2, it is characterised in that be with xylose or agriculture residual straw hydrolyzed solution as carbon source.
4. utilizing the method that serratia marcescens CGMCCNo.12714 described in claim 1 produces prodigiosin, its feature exists
In comprising the steps:
(1) primary-seed medium of serratia marcescens CGMCCNo.12714 is yeast extract 5g/L, peptone 10g/L, NaCl
10g/L, pH7.0, liquid amount 30mL, at 28-32 DEG C of condition 150-300rpm shake-flask culture 12-24h to OD600=1.0；
(2) within secondary seed medium is xylose 20g/L, peptone 25g/L, FeSO4·7H2O 0.23mM and CaCl2 16.68
MM, utilizes CaCO3PH is at 6.0-8.5 in control, and liquid amount 30mL, 3%-5% inoculum concentration, 28-32 DEG C of condition 150-300
Rpm lucifuge shake-flask culture 24-48h；
(3) the secondary seed culture fluid of inoculation 3%-5% ferments in 5L fermentation tank and produces prodigiosin, liquid amount 3L, primary carbon source
Within xylose or agriculture residual straw hydrolyzed solution total reducing sugars concentration 20g/L, peptone 25g/L, FeSO4·7H2O 0.23mM
And CaCl216.68mM, when culture medium middle peasant residual straw hydrolyzed solution total reducing sugars initial concentration is consumed to below 5g/L, single
Directly supplementing and concentrating agriculture residual straw hydrolyzed solution is original initial concentration to total reducing sugars concentration, and feed supplement number of times does not limits, and fermentation temperature is
28-32 DEG C, utilizing HCl and NaOH to control pH is 300-400rpm at 6.0-8.5, dissolved oxygen 40-60%, rotating speed, and lucifuge is sent out
Ferment 150-200h.
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| CN111621436A (en) * | 2020-06-02 | 2020-09-04 | 四川省农业科学院土壤肥料研究所 | Culture solution suitable for producing prodigiosin by Serratia marcescens XD1-B-1 |
| CN112080535A (en) * | 2020-09-26 | 2020-12-15 | 齐鲁工业大学 | Method for promoting synthesis and secretion of prodigiosin by using sodium humate |
| CN114561327A (en) * | 2022-03-31 | 2022-05-31 | 山东农业大学 | Cellulose degradation composite microbial inoculum and preparation method and application thereof |
| CN116731909A (en) * | 2023-05-08 | 2023-09-12 | 芝诺(苏州)生物科技有限公司 | Strain for high-yield prodigiosin and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111621436A (en) * | 2020-06-02 | 2020-09-04 | 四川省农业科学院土壤肥料研究所 | Culture solution suitable for producing prodigiosin by Serratia marcescens XD1-B-1 |
| CN111621436B (en) * | 2020-06-02 | 2022-05-13 | 四川省农业科学院土壤肥料研究所 | Culture solution suitable for producing prodigiosin by Serratia marcescens XD1-B-1 |
| CN112080535A (en) * | 2020-09-26 | 2020-12-15 | 齐鲁工业大学 | Method for promoting synthesis and secretion of prodigiosin by using sodium humate |
| CN114561327A (en) * | 2022-03-31 | 2022-05-31 | 山东农业大学 | Cellulose degradation composite microbial inoculum and preparation method and application thereof |
| CN114561327B (en) * | 2022-03-31 | 2023-09-12 | 山东农业大学 | Cellulose degradation composite microbial inoculant, and preparation method and application thereof |
| CN116731909A (en) * | 2023-05-08 | 2023-09-12 | 芝诺(苏州)生物科技有限公司 | Strain for high-yield prodigiosin and application thereof |
| CN116731909B (en) * | 2023-05-08 | 2024-01-26 | 芝诺(苏州)生物科技有限公司 | Strain for high-yield prodigiosin and application thereof |
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