CN112852891A - Artificial dual-bacterium system for producing mcl-PHA and application thereof - Google Patents
Artificial dual-bacterium system for producing mcl-PHA and application thereof Download PDFInfo
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- 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 100
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 90
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 claims abstract description 78
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims abstract description 77
- 241000588724 Escherichia coli Species 0.000 claims abstract description 64
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 50
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 50
- 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 46
- 239000008103 glucose Substances 0.000 claims abstract description 46
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 41
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 41
- 241000894006 Bacteria Species 0.000 claims abstract description 36
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 5
- 239000000194 fatty acid Substances 0.000 claims abstract description 5
- 229930195729 fatty acid Natural products 0.000 claims abstract description 5
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 38
- 230000004151 fermentation Effects 0.000 claims description 38
- 235000000346 sugar Nutrition 0.000 claims description 33
- 239000002609 medium Substances 0.000 claims description 26
- 239000000413 hydrolysate Substances 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 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 description 20
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 19
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 12
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- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 239000011734 sodium Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 229940059442 hemicellulase Drugs 0.000 claims description 4
- 108010002430 hemicellulase Proteins 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 229910021592 Copper(II) chloride Inorganic materials 0.000 claims description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 238000003501 co-culture Methods 0.000 abstract description 11
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- 238000001784 detoxification Methods 0.000 abstract description 6
- 235000016709 nutrition Nutrition 0.000 abstract description 4
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- 235000021588 free fatty acids Nutrition 0.000 description 14
- 239000002028 Biomass Substances 0.000 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
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- AUTALUGDOGWPQH-UBLOVXTBSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(2r,3s,4r)-2,3,4,5-tetrahydroxypentanal Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O AUTALUGDOGWPQH-UBLOVXTBSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
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- 235000019319 peptone Nutrition 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
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- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229920001221 xylan Polymers 0.000 description 2
- 150000004823 xylans Chemical class 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000193747 Bacillus firmus Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 101100061504 Escherichia coli cscB gene Proteins 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 241000209082 Lolium Species 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241000320117 Pseudomonas putida KT2440 Species 0.000 description 1
- 241001453296 Synechococcus elongatus Species 0.000 description 1
- 229940005348 bacillus firmus Drugs 0.000 description 1
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- 125000004432 carbon atom Chemical group C* 0.000 description 1
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- 208000015707 frontal fibrosing alopecia Diseases 0.000 description 1
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- 210000003709 heart valve Anatomy 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 235000015097 nutrients Nutrition 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
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- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
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- 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
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
Abstract
The invention relates to an artificial double-bacterium system for producing mcl-PHA and application thereof, wherein the artificial double-bacterium system consists of a recombinant bacterium E.coli delta p-m-a-e and a recombinant bacterium P.putida-acs. E.coli delta p-m-a-e is adopted to secrete acetic acid and extracellular fatty acid by taking xylose as a substrate; adopts P.putida-acs to utilize glucose as a main growth carbon source and utilizes acetic acid and FFA to synthesize medium-long-chain polyhydroxyalkanoate. Tasks such as substrate utilization, PHA synthesis and the like are distributed to different strains to be executed, the double bacteria form a mutual benefit symbiotic relationship based on 'nutrition supply' and 'detoxification', the metabolic function of a system is improved, the mcl-PHA yield obtained by double bacteria co-culture is 0.541g/L and is 3.9 times of that obtained by single bacteria pure culture, and the double bacteria system can realize the utilization of complex substrates, so that the substrate cost is reduced.
Description
Technical Field
The invention relates to an artificial double-bacterium system for producing mcl-PHA and application thereof, belonging to the field of microbiology mixed bacterium research.
Background
Polyhydroxyalkanoate (PHA) is a high-molecular polyester completely synthesized by microorganisms, and has various high value-added characteristics such as biodegradability and biocompatibility. The PHA products commercialized at present are mainly short chain polyhydroxyalkanoates (scl-PHA), and the further development and application of PHA are limited due to the high cost and small application range of the short chain polyhydroxyalkanoates (scl-PHA). mcl-PHA is a kind of PHA with 6-14 carbon atoms as monomer, and is synthesized mainly by Pseudomonas fluorescens (fluorescent Pseudomonas aeruginosa) under non-growth equilibrium condition. They have low crystallinity, low glass transition temperature, low tensile strength and high elongation at break and are elastomeric polymers. In addition, compared with scl-PHA, mcl-PHA has a more complex structure and is easier to be modified for use as a novel biomedical material such as a drug delivery material, a skin mask and a heart valve[1]. Therefore, the development of a process for synthesizing mcl-PHA while reducing the production cost thereof is expected to solve the problems of PHA at present.
The fermentable sugar such as glucose, xylose and the like from lignocellulose is a cheap renewable resource, and compared with the synthesis by using long-chain fatty acid as a substrate, the synthesis of mcl-PHA by using the fermentable sugar can effectively reduce the production cost, but the high-efficiency production cannot be realized. Currently, lignocellulose has been applied to PHA synthesis, mainly by converting lignocellulose into fermentable sugars (glucose and xylose) by various methods, and then biofermentation with sugar components to produce PHA. However, most of the studies were directed to PHB synthesis, and the synthesis of mcl-PHA has been rarely reported, Davis et al[2]mcl-PHA was synthesized from wild ryegrass hydrolysate treated in different ways using several different pseudomonas species. Although the yield of mcl-PHA is similar to that of the sugar mixture, the maximum yield is 0.3g/L, which is difficult to satisfyThe requirement of industrial production. According to previous studies, the main reason is that glucose and xylose are unrelated carbon sources of PHA structure, and different carbon sources have great influence on the biosynthesis of PHA. In general, carbon sources can be divided into "related" carbon sources { structures associated with PHA monomers, such as extracellular fatty acids (FFAs) } and "unrelated" carbon sources (structures not associated with PHA monomers, such as xylose and glucose)[3]。
The artificial mixed bacteria co-culture strategy becomes a new means of metabolic engineering and synthetic biology, and is widely used for overcoming the defects of pure culture process[4,5]. The application of mixed bacteria systems to PHA synthesis, Shalin, et al, has been studied[6]The PHB yield is improved by constructing a co-culture system consisting of Bacillus firmus NII 0830 and Lactobacillus delbrueckii NII 0925.
Etc. of[7]The method utilizes an artificial double-bacterium system consisting of blue algae and pseudomonas to react CO2Converted into mcl-PHA, and the yield reaches 156 mg/L. As can be seen, the mixed bacteria co-culture strategy has been widely applied to PHA synthesis. For pure culture of single bacteria and culture of natural mixed flora, the metabolic capability of the single bacteria is the main reason for limiting the yield of mcl-PHA, and complex substrates cannot be efficiently utilized, and the naturally formed or artificially domesticated mixed flora of microorganisms generally has the problems of long running period, low conversion efficiency, poor stability and controllability and the like. Therefore, by applying the method and theory of synthetic biology, the artificial double-bacterium system can be constructed to realize the task which can not be completed by pure culture microorganisms or improve the metabolic function of a multi-cell system. So far, no report is found on the research of synthesizing mcl-PHA by using an artificial mixed bacteria system and taking a glucose-xylose mixed carbon source and lignocellulose hydrolysate as substrates.
[1]Rai R,Keshavarz T,Roether J A,et al.Medium chain length polyhydroxyalkanoates,promising new biomedical materials for the future[J].Materials Science&Engineering R Reports,2011,72(3):29-47.
[2]Davis R,Kataria R,Cerrone F,et al.Conversion of grass biomass into fermentable sugars and its utilization for medium chain length polyhydroxyalkanoate(mcl-PHA)production by Pseudomonas strains[J].Bioresource Technology,2013,150(4):202-209.
[3] Chenqiang, widish yellow, microbial polyhydroxyalkanoate [ M ]. beijing: chemical industry press, 2014: 4-12.
[4]Jones J A,Wang X.Use of bacterial co-cultures for the efficient production of chemicals.[J].Curr Opin Biotechnol,2017,53:33-38.
[5]Zhang H,Wang X.Modular co-culture engineering,a new approach for metabolic engineering[J].Metabolic Engineering,2016,37:114-121.
[6]Shalin T,Sindhu R,Pandey A,et al.Production of poly-3-hydroxybutyrate from mixed culture[J].BIOLOGIA,2016,71(7):736-742.
[7]
H,Hobmeier K,Moos M,et al.Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB[J].Biotechnology for biofuels,2017,10(1):190.
Disclosure of Invention
In view of the above, the invention provides an artificial dual-bacterium system for producing mcl-PHA and application thereof, and verifies the feasibility thereof, and the specific technology is as follows:
the artificial double-bacterium system consists of a recombinant bacterium E.coli delta p-m-a-e and a recombinant bacterium P.putida-acs. The recombinant strain E.coli delta p-m-a-e utilizes xylose as a substrate to secrete acetic acid and extracellular fatty acid (FFA) by taking glucose and xylose mixed sugar as a carbon source; utilizing glucose as a main growth carbon source and utilizing acetic acid and FFA to synthesize mcl-PHA.
The double bacteria use the intermediate metabolite acetic acid to form a mutual beneficial symbiosis relationship based on 'nutrition supply-detoxification', namely P.putida-acs uses acetic acid secreted by E.coli delta p-m-a-e as a carbon source, and simultaneously relieves the inhibition of the acetic acid on the E.coli delta p-m-a-e. Then determining the initial inoculation ratio of the double bacteria, the relative inoculation time of the double bacteria and the nitrogen source concentration of the fermentation medium. And finally, applying the artificial dual-bacteria system to actual cellulose hydrolysate containing glucose and xylose.
The invention is specifically illustrated as follows:
an artificial dual-bacterium system for producing mcl-PHA; the artificial double-bacterium system consists of a recombinant bacterium E.coli delta p-m-a-e and a recombinant bacterium P.putida-acs.
The invention is used for producing an artificial double-bacterium system of mcl-PHA; the artificial double-bacterium system adopts recombinant bacterium E.coli delta p-m-a-e to secrete acetic acid and extracellular fatty acid (FFA) by using xylose as a substrate; the medium-long chain polyhydroxyalkanoate (mcl-PHA) is synthesized by using recombinant bacteria P.putida-acs and using acetic acid and FFA as main growth carbon sources.
The invention relates to a method for producing mcl-PHA by an artificial double-bacterium system; the method comprises the following steps:
(1) selecting single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs, respectively inoculating the single colonies to an LB liquid culture medium for culture so as to activate thalli; respectively sucking overnight cultures of the two strains into an M9 liquid culture medium, taking xylose as a carbon source for E.coli delta p-M-a-e culture, and taking glucose as a carbon source for P.putida-acs culture to respectively obtain seed solutions of E.coli delta p-M-a-e and P.putida-acs;
(2) inoculating the recombinant strain E.coli delta p-m-a-e obtained in the step (1) to a fermentation medium for fermentation; inoculating P.putida-acs into a fermentation medium for fermentation for producing mcl-PHA 12-24h after inoculation; the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are mixed according to the volume ratio of 0.5-2: 1, inoculation.
The fermentation medium of the step (2) comprises the following components in percentage by weight (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO4 0.24，NH4Cl, 1mL/L of trace element solution and pH of 7.0 +/-0.1; the carbon source is glucose and xylose mixed sugar, wherein the total sugar concentration is 20g/L, and the mass ratio of the glucose to the xylose is 1: 1; the concentration of the nitrogen source (ammonium chloride) is 1-4 g/L.
The formula of the trace elements (g/L1M HCl): FeSO4·7H2O 2.78，MnCl2·4H2O 1.98，CoCl2·6H2O 2.38，CaCl2·2H2O 1.47，CuCl2·2H2O 0.17，ZnSO4·7H2O 0.29。
The fermentation conditions in the step (2) are as follows: the fermentation temperature is 30 ℃, the filtered and sterilized air is supplied at the speed of 2vvm, and the Dissolved Oxygen (DO) is controlled to be more than 25 percent by automatically controlling the rotating speed; with automatic addition of 0.5M H2SO4And 1M KOH pH was controlled at 7.0. + -. 0.1.
The artificial double-bacterium system is applied to cellulose hydrolysate to produce mcl-PHA from mixed solution containing glucose and xylose.
The method for producing mcl-PHA by using the artificial dual-bacteria system to apply the enzymolysis of the corn straws as the cellulose hydrolysate, takes the corn straws pretreated by ammonia water as a substrate to carry out enzymolysis reaction, places the powder into a buffer solution containing citric acid to carry out reaction, and mixes the cellulose and the hemicellulase according to the ratio of 2: 1, adding in proportion; analyzing sugar components in the hydrolysate by using high performance liquid chromatography, wherein the sugar components in the hydrolysate are mainly glucose and xylose, and the ratio of the glucose to the xylose is 4.3: 1.
the 1M sodium citrate buffer solution: adding 210g of hydrated citric acid into 750mL of water, adding 50-60g of NaOH until the pH value is 4.3, and metering to 1L; the pH of this buffer was 4.8 when diluted to 0.05M.
The inoculation amount of the corresponding strains in the steps (1) and (2) can adopt the general technology in the prior art, and is usually 1-5%.
The recombinant bacterium E.coli delta p-m-a-e is constructed by a construction method of a genetic engineering bacterium which prefers to efficiently secrete acetic acid and FFA by utilizing xylose, according to the Chinese patent application number of 201810900753.6 applied by 2018-8-9 of Tianjin university and the name of the method for improving the acetic acid and FFA secretion capacity of escherichia coli and making escherichia coli prefer to utilize xylose.
The recombinant bacterium P.putida-acs is constructed according to the construction method of the genetic engineering bacterium for improving the acetic acid assimilation capability, which is disclosed as a method for improving the acetic acid assimilation capability of Pseudomonas putida KT2440 and applied to 2018-8-9 by Tianjin university under the patent application number of 201810900753.6.
The formula of the culture medium and the formula of the solution are as follows:
LB medium (g/L): 10.0 parts of peptone, 10.0 parts of NaCl, 5.0 parts of yeast extract powder and 7.0-7.2 parts of pH.
M9 mineral salts medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NH4Cl 2，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution and pH 7.0. The carbon source is added after converting glucose and xylose according to the required amount.
Glucose and xylose mother liquor: accurately weighing 100g of glucose and xylose, respectively dissolving in a blue-capped bottle containing 200mL of distilled water (final concentration of sugar is 500g/L), sterilizing with high pressure steam at 115 deg.C for 30min, cooling, and storing at 4 deg.C.
The application method of the artificial double-bacterium system in the production of mcl-PHA in actual cellulose hydrolysate is characterized in that the invention takes the corn stalk enzymolysis hydrolysate pretreated by ammonia water as the specific cellulose hydrolysate for producing mcl-PHA, and comprises the following steps:
(1) the enzymatic hydrolysis was carried out using corn stalks pretreated with ammonia water (water content: 7.57%, glucan content: 65.34; xylan content: 17.26%, insoluble lignin: 4.75%) as a substrate, the powder was placed in a 500mL Erlenmeyer flask containing a citric acid buffer solution (50mM, pH 4.8) and reacted at a concentration ratio of 10% (w/v), and cellulase and hemicellulase were reacted at a ratio of 2: 1 proportion, the reaction temperature is 50 ℃, the rotation speed is 200rpm, and the enzymolysis is carried out for 72 hours. And (3) after the enzymolysis is finished, putting the reaction liquid into a boiling water bath for 10min for enzyme deactivation, and then carrying out suction filtration to obtain hydrolysate. Analyzing sugar components in the hydrolysate by using high performance liquid chromatography, wherein the sugar components in the hydrolysate are mainly glucose and xylose, and the ratio of the glucose to the xylose is 4.3: 1.
(2) mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (1) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 0.5-2: 1 are sequentially inoculated to a fermentation medium for fermentation;
(3) and (3) inoculating the P.putida-acs to a fermentation medium 12-24 hours after inoculating the E.coli delta p-m-a-e in the step (2) for fermentation for producing mcl-PHA.
The fermentation medium in the steps (2) and (3) comprises the following specific components in percentage by weight (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO43，NaCl 0.5，MgSO4 0.24，NH4Cl, trace element solution 1mL/L, pH 7.0. The carbon source is the cellulose hydrolysate obtained by enzymolysis in the step (1), wherein the concentration of total sugar is 20g/L, and the concentration of the nitrogen source (ammonium chloride) is 4 g/L; meanwhile, reagent sugar is used for matching according to the same proportion and carrying out a control experiment.
Fermenting in a fermentation tank in the steps (2) and (3), controlling the fermentation temperature to be 30 ℃, supplying filtered and sterilized air at the speed of 2vvm, and controlling the Dissolved Oxygen (DO) to be more than 25% by automatically controlling the rotating speed; with automatic addition of 0.5M H2SO4And 1M KOH pH was controlled at 7.0. + -. 0.1.
The inoculation amounts of the corresponding strains in the steps (1) and (2) are all general techniques in the prior art.
And after fermentation for 48 hours, determining the mcl-PHA content in the fermentation liquor to be 0.434g/L, and showing that the constructed artificial dual-bacterium system has the capability of utilizing lignocellulose resources.
The invention has the advantages that:
the invention provides an artificial dual-bacterium system for producing mcl-PHA and application thereof, wherein an engineering P.putida-acs and E.coli delta p-m-a-e are utilized to construct the artificial dual-bacterium system, tasks such as substrate utilization, PHA synthesis and the like are distributed to different strains to be executed, and different strains are subjected to targeted design, transformation and reconstruction, the dual-bacterium system forms a mutual beneficial symbiotic relationship based on 'nutrition supply' and 'detoxification', the system metabolic function is improved, meanwhile, the single-bacterium metabolic overload is avoided, the mcl-PHA can be synthesized by utilizing a mixed carbon source of glucose and xylose, as shown in figure 2, the mcl-PHA yield obtained by co-culturing the dual-bacterium system is 0.541g/L and is 3.9 times of pure culturing of the single bacterium, in addition, the dual-bacterium system can realize the utilization of complex substrates, so as to reduce the substrate cost, the artificial dual-bacterium system can effectively synthesize the mcl-PHA by utilizing the straws of corn hydrolysate pretreated by ammonia water, the double-bacterium system is shown to have the potential of applying lignocellulose resources. Meanwhile, compared with other researches for synthesizing PHA by using renewable resources, the method has competitive advantages.
Drawings
FIG. 1 is a schematic diagram of the relationship between two bacteria
FIG. 2 E.coli Δ p-m-a-e and P.putida-acs comparison of mcl-PHA production in pure and co-culture
Under the conditions that the total sugar concentration is 20g/L and the glucose and the xylose are respectively 10g/L, the mcl-PHA yield obtained by double-bacterium co-culture under the optimal culture condition is 0.541g/L, and the yield is obviously improved compared with that obtained by single-bacterium pure culture.
FIG. 3 E.coli. DELTA.p-m-a-e and P.putida-acs in pure and cocultivation maximum biomass
FIG. 4 E.coli. DELTA.p-m-a-e and P.putida-acs changes in acetic acid and FFA concentrations in pure and co-cultures
In contrast to pure culture, acetic acid and FFA were hardly accumulated in the two-strain system, and both were used as a carbon source by P.putida-acs for synthesizing mcl-PHA, and the use of acetic acid released the inhibition of the growth of E.coli. DELTA.p-m-a-e by acetic acid.
FIG. 5 E.coli. DELTA.p-m-a-e and P.putida-acs changes in glucose concentration in pure and co-culture
In the case of pure E.coli. DELTA.p-m-a-e culture, glucose as the sole carbon source was consumed at 24h and in the case of pure P.putida-acs culture, glucose as the sole carbon source was consumed at 36h, whereas in the case of co-culture, glucose was still detectable at 60h, since P.putida-acs also utilized acetic acid and FFA secreted by E.coli. DELTA.p-m-a-e to grow and synthesize mcl-PHA.
FIG. 6 E.coli. DELTA.p-m-a-e and P.putida-acs changes xylose concentration in pure and co-culture
Under the condition of pure culture, the P.putida-acs can not utilize xylose at all, under the condition of co-culture, the xylose consumption in a double-bacterium system is faster, and the construction of the double-bacterium system promotes the utilization of the E.coli delta p-m-a-e to the xylose.
Detailed Description
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. The design of the dual-bacteria relationship is shown in figure 1, and the dual-bacteria system consists of a recombinant bacterium E.coli delta p-m-a-e and a recombinant bacterium P.putida-acs. The double-bacterium system takes glucose and xylose mixed sugar as a carbon source, the recombinant bacterium E.coli delta p-m-a-e prefers to utilize xylose to secrete extracellular long-chain Free Fatty Acid (FFA) and acetic acid, the recombinant bacterium P.putida-acs utilizes glucose as a growth carbon source, and simultaneously utilizes acetic acid and FFA to synthesize mcl-PHA. The double-bacterium system utilizes an intermediate metabolite acetic acid to construct a double-bacterium detoxification symbiotic relationship, namely the recombinant bacterium P.putida-acs utilizes the acetic acid secreted by the recombinant bacterium E.coli delta p-m-a-e as a carbon source, and simultaneously relieves the inhibition of the acetic acid on the recombinant bacterium E.coli delta p-m-a-e.
The invention is described in further detail below with reference to the following figures and specific examples:
1. culture medium formula and solution formula
LB medium (g/L): 10.0 parts of peptone, 10.0 parts of NaCl, 5.0 parts of yeast extract powder and 7.0-7.2 parts of pH.
M9 mineral salts medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NH4Cl 2，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution and pH 7.0. The carbon source is added after converting glucose and xylose according to the required amount.
Microelement formula (g/L1M HCl): FeSO4·7H2O 2.78，MnCl2·4H2O 1.98，CoCl2·6H2O 2.38，CaCl2·2H2O 1.47，CuCl2·2H2O 0.17，ZnSO4·7H2O 0.29。
Glucose and xylose mother liquor: accurately weighing 100g of glucose and xylose, respectively dissolving in a blue-capped bottle containing 200mL of distilled water (final concentration of sugar is 500g/L), sterilizing with high pressure steam at 115 deg.C for 30min, cooling, and storing at 4 deg.C.
1M sodium citrate buffer: to 750mL of water, 210g of citric acid hydrate was added, and 50 to 60g of NaOH was added to adjust the pH to 4.3, to make a volume of 1L. The pH of this buffer was 4.8 when diluted to 0.05M.
Example 1 the synergistic symbiotic relationship of E.coli. DELTA.p-m-a-e and P.putida-acs Artificial Dual bacterial System "Nutrition supply-detoxification
(1) Single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are picked and respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ for overnight culture under the condition of 220rpm so as to activate thalli. 1mL of the overnight culture of each of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli. DELTA.p-M-a-e culture was performed with xylose as a carbon source while P.putida-acs culture was performed with glucose as a carbon source.
(2) Mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (1) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 0.5: 1 in sequence, and p.putida-acs was inoculated 12h after e.coli Δ p-m-a-e inoculation.
The fermentation medium is M9 inorganic salt medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution and pH 7.0. Wherein the total sugar concentration is 20g/L, the ratio of glucose to xylose is 1: 1. the concentration of the nitrogen source (ammonium chloride) was 1 g/L.
Fermenting in 5L fermentation tank at 2L liquid loading rate, controlling fermentation temperature at 30 deg.C, supplying filtered and sterilized air at 2vvm rate, controlling Dissolved Oxygen (DO) at above 25% by automatically controlling rotation speed, and automatically adding 0.5M H2SO4And 1M KOH pH was controlled at 7.0. + -. 0.1. The fermentation tank culture experiment is carried out for 2 times of parallel experiments, and the mcl-PHA yield is 0.522g/L, which shows that the constructed artificial double-bacterium system can effectively utilize the glucose xylose mixed sugar in a laboratory as a carbon source to produce the mcl-PHA.
The change in sugar concentration and biomass during the culture were analyzed, and E.coli. DELTA.p-m-a-e OD after 48h of fermentation as shown in FIG. 3600Up to 6.27, P.putida-acs OD600Up to 8.56 by comparison of the maximum OD600It is known that in the two-strain system, the biomass of P.putida-acs and E.coli. DELTA.p-m-a-e were higher than those when each was cultured alone. The increase in biomass of E.coli. DELTA.p-m-a-e may be mainly due to the fact that the consumption of acetic acid by P.putida-acs via metabolism relieved the inhibition of its growth by acetic acid, and as shown in FIG. 4, acetic acid was hardly accumulated in the two-bacterium system as compared with the pure culture, whereas as can be seen from FIG. 6, the consumption of xylose was faster in the two-bacterium system, and the absence of residues also infers that E.coli. DELTA.p-m-a-e grew better.The increase in biomass of P.putida-acs is due to the increase in the available carbon source in the co-cultivation system, as shown in FIG. 5, in the pure cultivation case, glucose as the sole carbon source is consumed at 24h, making it difficult to continue increasing the biomass, while in the co-cultivation system, P.putida-acs can also grow and synthesize mcl-PHA by using acetic acid and FFA secreted by E.coli Δ p-m-a-e. These results demonstrate that the dual bacteria use the intermediate metabolite, acetic acid, to form a "nutrient supply-detoxification" based mutualistic symbiosis relationship.
Example 2 E.coli. DELTA.p-m-a-e and P.putida-acs Artificial Dual bacteria System for production of mcl-PHA Using laboratory Mixed sugar
(1) Single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are picked and respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ for overnight culture under the condition of 220rpm so as to activate thalli. 1mL of the overnight culture of each of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli. DELTA.p-M-a-e culture was performed with xylose as a carbon source while P.putida-acs culture was performed with glucose as a carbon source.
(2) Mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (1) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 2: 1 in sequence, and p.putida-acs 15h after e.coli Δ p-m-a-e inoculation.
The fermentation medium in the step (2) is M9 inorganic salt medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution and pH 7.0. Wherein the total sugar concentration is 20g/L, the ratio of glucose to xylose is 1: 1. the concentration of the nitrogen source (ammonium chloride) was 3 g/L.
The fermenter parameter control in step (2) was the same as in example 1. After fermentation for 48 hours, the mcl-PHA yield is 0.492g/L, and compared with the mcl-PHA yield produced by other mixed floras, the yield is remarkably improved.
Example 3 production of mcl-PHA Using A laboratory Mixed sugar in an E.coli Δ p-m-a-e and P.putida-acs Artificial Dual bacteria System
(1) Single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are picked and respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ for overnight culture under the condition of 220rpm so as to activate thalli. 1mL of the overnight culture of each of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli. DELTA.p-M-a-e culture was performed with xylose as a carbon source while P.putida-acs culture was performed with glucose as a carbon source.
(2) Mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (1) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 1: 1 in sequence, and p.putida-acs was inoculated 12h after e.coli Δ p-m-a-e inoculation.
The fermentation medium in the step (2) is M9 inorganic salt medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution and pH 7.0. Wherein the total sugar concentration is 20g/L, the ratio of glucose to xylose is 1: 1. the concentration of the nitrogen source (ammonium chloride) was 2 g/L.
The fermenter parameter control in step (2) was the same as in example 1. As shown in figure 2, after 48 hours of fermentation, the yield of mcl-PHA co-cultured by double bacteria is 0.541g/L, and the yield is obviously improved compared with that of single bacteria pure culture.
Example 4 E.coli Δ p-m-a-e and P.putida-acs artificial dual bacteria System Using Mixed fermentation of cellulose hydrolysate to produce mcl-PHA
(1) The enzymatic hydrolysis was carried out using corn stalks pretreated with ammonia water (water content: 7.57%, glucan content: 65.34; xylan content: 17.26%, insoluble lignin: 4.75%) as a substrate, the powder was placed in a 500mL Erlenmeyer flask containing a citric acid buffer solution (50mM, pH 4.8) and reacted at a concentration ratio of 10% (w/v), and cellulase and hemicellulase were reacted at a ratio of 2: 1 proportion, the reaction temperature is 50 ℃, the rotation speed is 200rpm, and the enzymolysis is carried out for 72 hours. And (3) after the enzymolysis is finished, putting the reaction liquid into a boiling water bath for 10min for enzyme deactivation, and then carrying out suction filtration to obtain hydrolysate. Analyzing sugar components in the hydrolysate by using high performance liquid chromatography, wherein the sugar components in the hydrolysate are mainly glucose and xylose, and the ratio of the glucose to the xylose is 4.3: 1.
(2) single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are picked and respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ for overnight culture under the condition of 220rpm so as to activate thalli. 1mL of the overnight culture of each of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli. DELTA.p-M-a-e culture was performed with xylose as a carbon source while P.putida-acs culture was performed with glucose as a carbon source.
(3) Mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (2) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 1: 1 in sequence, and p.putida-acs in 24h after e.coli Δ p-m-a-e inoculation.
The fermentation medium in the step (3) is M9 inorganic salt medium (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO40.24, 1mL/L of trace element solution, pH 7.0, 4g/L of nitrogen source (ammonium chloride), fermentation by taking cellulose hydrolysate obtained by enzymolysis as a carbon source, wherein the total sugar concentration is 20g/L, and meanwhile, proportioning is carried out by using reagent sugar in the same proportion and a control experiment is carried out.
The fermenter parameter control was the same as in example 1. After fermentation for 48 hours, the yield of mcl-PHA produced by fermentation with hydrolysate as a carbon source is 0.434g/L, which shows that the constructed artificial double-bacterium system has the capability of utilizing lignocellulose resources.
While the invention has been described in further detail with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit of the invention, and such changes and modifications are to be considered within the scope of the invention.
Claims (9)
1. An artificial dual-bacterium system for producing mcl-PHA; the artificial double-bacterium system is characterized by consisting of a recombinant bacterium E.coli delta p-m-a-e and a recombinant bacterium P.putida-acs.
2. An artificial dual bacteria system for the production of mcl-PHA as claimed in claim 1; the method is characterized in that the artificial dual-bacterium system adopts recombinant bacterium E.coli delta p-m-a-e to secrete acetic acid and extracellular fatty acid (FFA) by using xylose as a substrate; the medium-long chain polyhydroxyalkanoate (mcl-PHA) is synthesized by using recombinant bacteria P.putida-acs and using acetic acid and FFA as main growth carbon sources.
3. A method for producing mcl-PHA by the artificial dual bacterium system of claim 1 or 2; the method is characterized by comprising the following steps:
(1) selecting single colonies of the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs, respectively inoculating the single colonies to an LB liquid culture medium for culture so as to activate thalli; respectively sucking overnight cultures of the two strains into an M9 liquid culture medium, taking xylose as a carbon source for E.coli delta p-M-a-e culture, and taking glucose as a carbon source for P.putida-acs culture to respectively obtain seed solutions of E.coli delta p-M-a-e and P.putida-acs;
(2) inoculating the recombinant strain E.coli delta p-m-a-e obtained in the step (1) to a fermentation medium for fermentation; inoculating P.putida-acs into a fermentation medium for fermentation for producing mcl-PHA 12-24h after inoculation; the recombinant strain E.coli delta p-m-a-e and the recombinant strain P.putida-acs are mixed according to the volume ratio of 0.5-2: 1, inoculation.
4. The method of claim 3, wherein the fermentation medium of step (2) has a composition (g/L): na (Na)2HPO4·7H2O 12.8，KH2PO4 3，NaCl 0.5，MgSO4 0.24，NH4Cl, 1mL/L of trace element solution and pH of 7.0 +/-0.1; the carbon source is glucose and xylose mixed sugar, wherein the total sugar concentration is 20g/L, and the mass ratio of the glucose to the xylose is 1: 1; the concentration of the nitrogen source (ammonium chloride) is 1-4 g/L.
5. The process according to claim 4, wherein the trace element formulation (g/L1M HCl): FeSO4·7H2O 2.78，MnCl2·4H2O 1.98，CoCl2·6H2O 2.38，CaCl2·2H2O 1.47，CuCl2·2H2O 0.17，ZnSO4·7H2O 0.29。
6. The method as set forth in claim 3, wherein the fermentation conditions in step (2) are: the fermentation temperature is 30 deg.C, the filtered and sterilized air is supplied at 2vvm rate, and the Dissolved Oxygen (DO) is controlled to above 25% by automatically controlling the rotation speed(ii) a With automatic addition of 0.5M H2SO4And 1M KOH pH was controlled at 7.0. + -. 0.1.
7. The application of the artificial dual-bacteria system of claim 1 in producing mcl-PHA from cellulose hydrolysate which is a mixed solution containing glucose and xylose.
8. The method for producing mcl-PHA by using the artificial dual-bacteria system as claimed in claim 1 and using corn stalks as substrates to perform enzymolysis reaction, placing the powder in a buffer solution containing citric acid to perform reaction, and mixing cellulase and hemicellulase according to the ratio of 2: 1, adding in proportion; analyzing sugar components in the hydrolysate by using high performance liquid chromatography, wherein the sugar components in the hydrolysate are mainly glucose and xylose, and the ratio of the glucose to the xylose is 4.3: 1.
9. the method of claim 8, wherein the buffer is 1M sodium citrate buffer: adding 210g of hydrated citric acid into 750mL of water, adding 50-60g of NaOH until the pH value is 4.3, and metering to 1L; the pH of this buffer was 4.8 when diluted to 0.05M.
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| CN113502242A (en) * | 2021-06-26 | 2021-10-15 | 北京大学深圳研究院 | Bacillus firmus and application thereof |
| CN115725667A (en) * | 2022-09-05 | 2023-03-03 | 天津大学 | Artificial dual-bacterium system for producing mcl-PHA (polyhydroxyalkanoate) by utilizing lignocellulose hydrolysate and application thereof |
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