CN106148255B - Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol - Google Patents
Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol Download PDFInfo
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- CN106148255B CN106148255B CN201510167062.6A CN201510167062A CN106148255B CN 106148255 B CN106148255 B CN 106148255B CN 201510167062 A CN201510167062 A CN 201510167062A CN 106148255 B CN106148255 B CN 106148255B
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 59
- 241000894006 Bacteria Species 0.000 title claims abstract description 56
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 25
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 24
- 150000007524 organic acids Chemical class 0.000 title claims abstract description 21
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 98
- 241000588748 Klebsiella Species 0.000 claims abstract description 78
- 108010092060 Acetate kinase Proteins 0.000 claims abstract description 18
- 230000037361 pathway Effects 0.000 claims abstract description 15
- 108020003285 Isocitrate lyase Proteins 0.000 claims abstract description 13
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims abstract description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 5
- 108010042687 Pyruvate Oxidase Proteins 0.000 claims abstract 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 62
- 238000000855 fermentation Methods 0.000 claims description 42
- 230000004151 fermentation Effects 0.000 claims description 42
- 108090000854 Oxidoreductases Proteins 0.000 claims description 31
- 235000019441 ethanol Nutrition 0.000 claims description 31
- 239000004310 lactic acid Substances 0.000 claims description 31
- 235000014655 lactic acid Nutrition 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 23
- 101710088194 Dehydrogenase Proteins 0.000 claims description 22
- BWLBGMIXKSTLSX-UHFFFAOYSA-N 2-hydroxyisobutyric acid Chemical compound CC(C)(O)C(O)=O BWLBGMIXKSTLSX-UHFFFAOYSA-N 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 16
- 238000013459 approach Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 230000002779 inactivation Effects 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Chinese gallotannin Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims description 4
- 239000012531 culture fluid Substances 0.000 claims description 4
- 235000009508 confectionery Nutrition 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 230000003519 ventilatory effect Effects 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
- 238000003786 synthesis reaction Methods 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 150000001298 alcohols Chemical class 0.000 abstract description 5
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 abstract description 3
- 229940035437 1,3-propanediol Drugs 0.000 abstract description 3
- 235000005985 organic acids Nutrition 0.000 abstract description 3
- 229920000166 polytrimethylene carbonate Polymers 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 abstract 1
- 108010012901 Succinate Dehydrogenase Proteins 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 238000011144 upstream manufacturing Methods 0.000 description 44
- 238000012408 PCR amplification Methods 0.000 description 33
- 108090000623 proteins and genes Proteins 0.000 description 31
- 239000012634 fragment Substances 0.000 description 30
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 27
- 235000011187 glycerol Nutrition 0.000 description 25
- 101150041530 ldha gene Proteins 0.000 description 22
- 230000029087 digestion Effects 0.000 description 20
- 239000000203 mixture Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 19
- 101150006213 ackA gene Proteins 0.000 description 16
- 101150060030 poxB gene Proteins 0.000 description 16
- 101150075213 frdA gene Proteins 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 12
- 101150094017 aceA gene Proteins 0.000 description 12
- 101100398785 Streptococcus agalactiae serotype V (strain ATCC BAA-611 / 2603 V/R) ldhD gene Proteins 0.000 description 11
- 101100386830 Zymomonas mobilis subsp. mobilis (strain ATCC 31821 / ZM4 / CP4) ddh gene Proteins 0.000 description 11
- 238000003209 gene knockout Methods 0.000 description 11
- 101150026107 ldh1 gene Proteins 0.000 description 11
- 101100462488 Phlebiopsis gigantea p2ox gene Proteins 0.000 description 10
- 239000007787 solid Substances 0.000 description 9
- 101100242035 Bacillus subtilis (strain 168) pdhA gene Proteins 0.000 description 8
- 206010010144 Completed suicide Diseases 0.000 description 8
- 101100123255 Komagataeibacter xylinus aceC gene Proteins 0.000 description 8
- 101100433987 Latilactobacillus sakei subsp. sakei (strain 23K) ackA1 gene Proteins 0.000 description 8
- 101100134871 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aceE gene Proteins 0.000 description 8
- SCJNCDSAIRBRIA-DOFZRALJSA-N arachidonyl-2'-chloroethylamide Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCCCl SCJNCDSAIRBRIA-DOFZRALJSA-N 0.000 description 8
- 101150070136 axeA gene Proteins 0.000 description 8
- 230000006801 homologous recombination Effects 0.000 description 7
- 238000002744 homologous recombination Methods 0.000 description 7
- 235000002639 sodium chloride Nutrition 0.000 description 7
- 241000607142 Salmonella Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 102000003960 Ligases Human genes 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 101100378108 Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh) icl2 gene Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- -1 antifreeze.Currently Substances 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000001384 succinic acid Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 1
- 241000588923 Citrobacter Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 241001609931 bacterium 20 Species 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 239000002816 fuel additive Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000003317 industrial substance Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920003051 synthetic elastomer Polymers 0.000 description 1
- 239000005061 synthetic rubber Substances 0.000 description 1
- ODBLHEXUDAPZAU-UHFFFAOYSA-N threo-D-isocitric acid Natural products OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an engineering bacterium lacking in an organic acid production way and application thereof in coproduction of 1, 3-propylene glycol, 2, 3-butanediol and ethanol. The invention inactivates the lactate dehydrogenase gene of the lactate synthesis pathway of the Klebsiella, the acetate kinase gene and the pyruvate oxidase gene of the acetate synthesis pathway, and the fumarate reductase gene and the isocitrate lyase gene of the succinate synthesis pathway, thereby reducing the production of organic acids when the Klebsiella metabolizes glycerol, improving the yield and conversion rate of alcohols such as 1, 3-propanediol, 2, 3-butanediol and ethanol, and realizing the co-production of 1, 3-propanediol, 2, 3-butanediol and ethanol. The yield of the organic acid is greatly reduced, and the extraction process of the alcohol can be simplified. The invention can improve the production efficiency of high-value products produced by converting glycerol with microorganisms, reduce the production cost and has important application value.
Description
Technical field
The invention belongs to technical field of biochemical industry, in particular to the one plant engineering bacteria for producing organic acid approach missing and its
Application in coproduction 1,3- propylene glycol, 2,3- butanediol and ethyl alcohol.
Background technique
1,3-PD is a kind of important industrial chemicals, can be used for the industries such as solvent, lubricant, antifreeze.Currently, 1,
3-propanediol is mainly used for synthesizing polypropylene terephthalate (PTT), and the latter is a kind of biodegradable haveing excellent performance
Polyester material, have good ductility and washing property, weaving, in terms of have broad application prospects.Biology
Method fermenting carbohydrate or glycerol production 1,3-PD are a kind of 1,3-PD production methods efficiently, environmentally friendly, by increasingly
More concerns.Klebsiella (Klebsiella), citric acid Pseudomonas (Citrobacter) and fusobacterium
(Clostridium) etc. bacteriums have been used for fermentation glycerol production 1,3- propylene glycol (Saxena et al.,
Biotechnol.Adv.,2009,27,895–913)。
2,3-butanediol is a kind of widely used platform chemicals, has weight in multiple fields such as fuel, chemical industry, food
The purposes wanted.2,3-butanediol calorific value with higher, can be used as fuel additive；1,4-butanediol can be replaced, polyester is used for
With the synthesis of polyurethane；Its dehydration product methyl ethyl ketone is that one kind is widely used in the industries such as coating, lubricant, fuel, fragrance
Solvent；Its dehydration product 1,3-butadiene can be used to synthetic rubber monomer, be a kind of important organic original of basic petrochemical
Material；Its high level derivative 3-Hydroxybutanone and diacetyl are widely used in the industries such as food, cosmetics, fragrance.Microbial fermentation
Method production 2,3-butanediol is widely studied, and the bacterium in nature with 2,3-butanediol production capacity is mainly wrapped
It includes: Klebsiella (Klebsiella), bacillus (Bacillus), Serratia (Serratia) and enterobacteria
Belong to (Jietal., Biotechnol.Adv., 2011,29,351-364.) such as (Enterobacter).
Ethyl alcohol is to study one of most commonly used bio-fuel at present, raw using microbial fermentations such as yeast, Escherichia coli
Generation ethyl alcohol, it has also become the hot spot in regenerated biological energy field.Traditional technique using starch sugar fermentative production of ethanol,
It is at high cost, and grain is striven with people.Therefore, development and utilization non-grain raw material such as cellulosic hydrolysate, sweet is focused in more and more researchs
Oil production ethyl alcohol.
Currently, 1,3-PD, 2,3-butanediol and the respective bioanalysis production technology of ethyl alcohol are widely ground
Study carefully, but the report of these three important chemicals of coproduction not yet.Klebsiella can produce 1,3- the third two when being metabolized glycerol
The alcohols such as alcohol 2,3-butanediol and ethyl alcohol, while can also produce the organic acids such as lactic acid, acetic acid and succinic acid.Lack Klebsiella
The route of synthesis of middle organic acid, the production of organic acid when being expected to reduce Klebsiella fermentation glycerol, to improve the life of alcohols
Efficiency and conversion ratio are produced, realizes fermentation glycerol coproduction 1,3-PD, 2,3-butanediol and ethyl alcohol.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide one plant produce organic acid approach missing engineering bacteria and
Its application method in coproduction 1,3- propylene glycol, 2,3- butanediol and ethyl alcohol.
Of the invention one plant produces the engineering bacteria of organic acid approach missing, which is characterized in that Klebsiella wild-type strain
Lactic acid producing approach, produce acetate pathway and produce succinate pathway missing, which is named as Klebsiella XP-5Klebsiella
Sp.XP-5 is preserved in China typical culture collection center on March 6th, 2015, address: Wuhan, China Wuhan University,
Deposit number are as follows: CCTCC NO M 2015092.
The lacking lactic acid production path is inactivation lactic acid dehydrogenase gene；The production acetate pathway missing is that inactivation acetic acid swashs
Enzyme gene and acetonic acid oxidase gene；The production succinate pathway missing is inactivation fumaric reductase gene and isocitric acid
Lyase gene.
The lactic acid dehydrogenase gene sequence such as SEQ ID NO.1；The Acetokinase gene sequence such as SEQ ID
NO.2；The acetonic acid oxidase gene sequence such as SEQ ID NO.3；The fumaric reductase gene order such as SEQ ID
NO.4；The isocitrate lyase gene order such as SEQ ID NO.5.
The lactic acid dehydrogenase gene of the inactivation Klebsiella, Acetokinase gene, acetonic acid oxidase gene, rich horse
Sour reductase gene and isocitrate lyase gene, are realized by gene knockout, preferably carry out base by the following method
Because knocking out:
(1) homeologous sequence of the lactic acid dehydrogenase gene of PCR amplification Klebsiella in genome middle and upper reaches and downstream
Column, it are connected with suicide vector, then again in introductive crossing donor bacterium；
(2) suicide for carrying lactic acid dehydrogenase gene upstream and downstream homeologous sequence for obtaining step (1) carries
The donor bacterium of body and Klebsiella carry out double parents, lactic acid dehydrogenase gene are knocked out using homologous recombination, after screening
Obtain the Klebsiella that lactic acid dehydrogenase gene is knocked；
(3) Acetokinase gene of PCR amplification Klebsiella genome middle and upper reaches and downstream homeologous sequence,
It is connected with suicide vector, then again in introductive crossing donor bacterium；
(4) suicide vector for carrying Acetokinase gene upstream and downstream homeologous sequence for obtaining step (3)
Donor bacterium and step (2) Klebsiella for being knocked of lactic acid dehydrogenase gene for obtaining carry out double parents, using same
Source recombination knocks out Acetokinase gene, the Cray that acquisition lactic acid dehydrogenase gene and Acetokinase gene are all knocked after screening
Primary Salmonella；
(5) homeologous of the acetonic acid oxidase gene of PCR amplification Klebsiella in genome middle and upper reaches and downstream
It is connected by sequence with suicide vector, then again in introductive crossing donor bacterium；
(6) suicide for carrying acetonic acid oxidase gene upstream and downstream homeologous sequence for obtaining step (5)
The Klebsiella that the lactic acid dehydrogenase gene and Acetokinase gene that the donor bacterium of carrier and step (4) obtain are knocked carries out
Double parents, knock out acetonic acid oxidase gene using homologous recombination, lactic acid dehydrogenase gene are obtained after screening, acetic acid swashs
The Klebsiella that enzyme gene and acetonic acid oxidase gene are all knocked；
(7) homeologous of the fumaric reductase gene of PCR amplification Klebsiella in genome middle and upper reaches and downstream
It is connected by sequence with suicide vector, then again in introductive crossing donor bacterium；
(8) suicide for carrying fumaric reductase upstream region of gene and downstream part homologous sequence for obtaining step (7)
Lactic acid dehydrogenase gene, Acetokinase gene and the acetonic acid oxidase gene that the donor bacterium of carrier obtains with step (6) are struck
The Klebsiella removed carries out double parents, knocks out fumaric reductase gene using homologous recombination, cream is obtained after screening
The Cray primary that dehydrogenase gene, Acetokinase gene, acetonic acid oxidase gene and fumaric reductase gene are all knocked
Salmonella；
(9) the isocitrate lyase gene of PCR amplification Klebsiella is same in the part in genome middle and upper reaches and downstream
It is connected by source sequence with suicide vector, then again in introductive crossing donor bacterium；
(10) by step (9) obtain carry isocitrate lyase upstream region of gene and downstream part homologous sequence from
Lactic acid dehydrogenase gene, Acetokinase gene, acetonic acid oxidase gene and the richness that the donor bacterium and step (8) for killing carrier obtain
The Klebsiella that horse acid reductase gene is knocked carries out double parents, knocks out isocitrate lyase using homologous recombination
Gene obtains lactic acid dehydrogenase gene, Acetokinase gene, acetonic acid oxidase gene, fumaric reductase base after screening
The Klebsiella that cause and isocitrate lyase gene are all knocked.
The PCR amplification Klebsiella lactic acid dehydrogenase gene upstream portion homologous sequence of the step (1), be with gram
The genomic DNA of the primary Salmonella of thunder is template, with primer ldhA-1:ATCGGAATTCAGGGTATTGAGCTGGGCGTC and primer
The primer of ldhA-2:ATTCAAGCTTCGAAACCTGTCCGAACGCCA composition obtains upstream sequence after carrying out PCR amplification；To draw
Object ldhA-3:ATTAAAGCTTCCGTTGGCGGTTTTGGCAGT and primer ldhA-4:
The primer of TCTACCCGGGTTTTCAGCCGCTTTCTCTCT composition obtains downstream sequence after carrying out PCR amplification.
The PCR amplification Klebsiella Acetokinase gene upstream and downstream homeologous sequence of the step (3), be with
The genomic DNA of Klebsiella is template, with primer ackA-1:ATCGGAATTCTAGCGGGTGGCACGAATAAT and primer
The primer of ackA-2:ATTAGGATCCGCTACCGCAGTTCAGAACCA composition obtains upstream sequence after carrying out PCR amplification；To draw
Object ackA-3:GCAAGGATCCCTATACCATCGCACTGACCG and primer ackA-4:
The primer of TCCCCCCGGGCGAGACAAAAGACTTTCATC composition obtains downstream sequence after carrying out PCR amplification.
The PCR amplification Klebsiella acetonic acid oxidase gene upstream and downstream homeologous sequence of the step (5),
Be using the genomic DNA of Klebsiella as template, with primer poxB-1:GCATGAATTCTTTCGCTGCCACTTTATCCA with
The primer of primer poxB-2:ATTAGGATCCGGCGAAAACCAACTGGCTCA composition obtains upstream sequence after carrying out PCR amplification；
With primer poxB-3:ATGCGGATCCACGGTCTGCTTCATGATCTC and primer poxB-4:
The primer of CGTACTGCAGATCTAAGCCGACCATCAGCC composition obtains downstream sequence after carrying out PCR amplification.
The PCR amplification Klebsiella fumaric reductase upstream region of gene and downstream part homologous sequence of the step (7),
Be using the genomic DNA of Klebsiella as template, with primer frdA-1:ATTCGAATTCGGTCTGCTTGTTTGGCCCCT with
The primer of primer frdA-2:CATGCATATGCCGTCTGGAATATGGCGATG composition obtains upstream sequence after carrying out PCR amplification；
With primer frdA-3:ATTCCATATGCGTATGGCTGCGCATCGGAT and primer frdA-4:
The primer of TAACCTGCAGCGGTAAAGAAACGGCGGATT composition obtains downstream sequence after carrying out PCR amplification.
The homologous sequence of PCR amplification Klebsiella isocitrate lyase upstream region of gene and downstream part of the step (9)
Column, are using the genomic DNA of Klebsiella as template, with primer aceA-1:
ATGCGAATTCGAACGCACGGAAGCCGGAAT and primer aceA-2:ATCGAAGCTTGCGGGTTCGTCCATTCTTTG is formed
Primer carry out PCR amplification after obtain upstream sequence；With primer aceA-3:ATCGAAGCTTTTGAGAAAGTGCAGCAGCCG with
The primer of primer aceA-4:ATGGCTGCAGAGGCCCACGTGGTGATCGTA composition obtains downstream sequence after carrying out PCR amplification.
The suicide vector be suicide vector pKR6K (such as Fig. 1, Wang et al., J.Biol.Chem.2014,289:
6080-6090).In the derivative vector introductive crossing F+strain of the suicide vector, heat shock method, electrotransformation can be passed through
The conventional methods conversion hybridization donor bacterium such as method, engagement conversion method.
The hybridization donor bacterium is Escherichia coli S17-1 (λ pir).
The present invention also provides produce the engineering bacteria of organic acid approach missing in coproduction 1,3- propylene glycol, 2,3- butanediol and second
The step of application method in alcohol, this method, is as follows:
(1) seed culture: Klebsiella XP-5CCTCC M 2015092 is selected, is aseptically seeded to and is equipped with
It in the 500mL triangular flask of glycerol fermentation culture medium, 30-37 DEG C, cultivates 8-15 hours in 100-200rpm shaking table, obtains seed training
Nutrient solution.
(2) seed culture fluid obtained in step (1) fermented and cultured: is seeded to the hair of the 5L equipped with glycerol fermentation culture medium
In fermentation tank, fed batch fermentation is carried out, inoculum concentration is 0.5-5% (v/v), and ventilatory capacity 0.5-2.0vvm, speed of agitator is
100-300rpm, fermentation temperature are 30-37 DEG C, and in fermentation process, adjusting fermentation liquid pH using aqueous slkali is 6.0-7.0.When sweet
When glycerol depletion in oily fermentation medium, by adding the glycerite of 500-700g/L into fermentor, fermentation liquid is controlled
Middle glycerol concentration is 5-30g/L.When the concentration of 1,3-PD, 2,3-butanediol and ethyl alcohol in fermentation liquid no longer increases, stop
Only ferment.
Contain glycerol as carbon source in the glycerol fermentation culture medium.
The organic acid route of synthesis of Klebsiella is included lactic acid route of synthesis, acetic acid route of synthesis and amber by the present invention
The key gene of sour route of synthesis carries out gene knockout, thus reduce the production of organic acid when Klebsiella is metabolized glycerol,
The yield and conversion ratio for improving alcohols such as 1,3-PD, 2,3-butanediol and ethyl alcohol realize 1,3-PD, 2,3- fourth
The coproduction of two pure and mild ethyl alcohol.Production of organic acids is greatly reduced, and can simplify the extraction process of alcohols.Micro- life can be improved in the present invention
Object glycerine converting produces the production efficiency of high level product, reduces production cost, has important application value.
Detailed description of the invention
The physical map of Fig. 1 suicide vector pKR6K.
Fig. 2 lactic acid producing, the engineering bacteria XP-5 fermentation glycerol coproduction 1,3- propylene glycol of acetic acid and succinate pathway missing, 2,3-
The conditional curve of butanediol and ethyl alcohol.
Specific embodiment
Technology contents of the invention are described further below with reference to embodiment: following embodiments be it is illustrative,
It is not restrictive, cannot be limited the scope of protection of the present invention with following embodiments.Experiment as used in the following examples
Method is conventional method unless otherwise specified.The materials, reagents and the like used in the following examples, unless otherwise specified,
It obtains from commercial channels.
Embodiment one: the knockout of lactic acid dehydrogenase gene ldhA in Klebsiella
(1) clone of lactic acid dehydrogenase gene ldhA upstream and downstream homeologous sequence
According to the genome sequence design primer of Klebsiella ATCC 25955, on PCR lactic acid dehydrogenase gene ldhA
Trip and downstream part homologous sequence.Using the genomic DNA of Klebsiella ATCC 25955 as template, with primer ldhA-1:
ATCGGAATTCAGGGTATTGAGCTGGGCGTC and primer ldhA-2:ATTCAAGCTTCGAAACCTGTCCGAACGCCA is formed
Primer carry out PCR amplification, obtain the homeologous sequence of the upstream ldhA；With primer ldhA-3:
ATTAAAGCTTCCGTTGGCGGTTTTGGCAGT and primer ldhA-4:TCTACCCGGGTTTTCAGCCGCTTTCTCTCT is formed
Primer carry out PCR amplification, obtain the homeologous sequence in the downstream ldhA.PCR amplification condition are as follows: 95 DEG C 5 minutes；95℃30
Second, 60 DEG C 30 seconds, 72 DEG C 1 minute, totally 30 circulation；72 DEG C 5 minutes.After reaction, pcr amplification product is carried out by PCR
1.0% agarose gel electrophoresis recycles and purifies, obtains target fragment.
(2) suicide vectorBuilding
Using the homologous fragment of restriction enzyme EcoRI and HindIII digestion ldhA upstream region of gene, HindIII is used
With the homologous fragment of XmaI digestion ldhA downstream of gene, using EcoRI and XmaI digestion pKR6K carrier, by digestion products and
PKR6K carrier is recycled and is purified, using T4 ligase, by the homologous fragment of ldhA upstream region of gene, the homologous fragment in downstream
It is connected with pKR6K carrier, connection product is transferred to Escherichia coli S17-1 (λ pir), screened positive transformant, carried
The suicide vector of ldhA upstream region of gene and downstream part homologous fragment
(3) building for the Klebsiella that lactic acid dehydrogenase gene ldhA is knocked out
It is carried what is obtained in step (2)Escherichia coli S17-1 (λ pir) (donor bacterium) with gram
The primary Salmonella ATCC25955 (recipient bacterium) of thunder carries out double parents, makesOn ldhA upstream region of gene and downstream
Homeologous sequence and Klebsiella genome occur homologous recombination, to make the ldhA gene delection in Klebsiella
Inactivation, to obtain the Klebsiella of ldhA gene knockout.Method particularly includes:
A. the donor bacterium after inoculation activation and recipient bacterium are into 5mLLB culture medium respectively, in 37 DEG C of shaking tables, 200rpm culture
2-3 hours, when donor bacterium and recipient bacterium grow into OD simultaneously620nmFor 0.5-0.8；5mL donor bacterium bacterium solution is centrifuged, sterile physiological
Salt is washed twice；1mL recipient bacterium bacterium solution is centrifuged, sterile saline is washed twice；By the thallus of above-mentioned donor bacterium and recipient bacterium
One, which shares 100 μ L sterile salines, is resuspended, and by re-suspension liquid, all drop among LB solid medium tablets, put by plate front
Set, 37 DEG C culture 12-18 hours.
B. the bacterium colony in step a in LB solid medium tablets is scraped with sterile saline and scraper, sterile physiological
Salt is washed twice, is suitably diluted, and the M9 solid medium tablets that joined 50 μ g/mL kanamycins, 37 DEG C of trainings are coated on
It supports 24-36 hours.
C. the single bacterium grown in M9 solid medium tablets in picking step b drops down onto the 5mL that 50 μ g/mL kanamycins are added
In LB culture medium, 37 DEG C, 200rpm is cultivated 12 hours.(not adding card, that is mould into fresh 5mL LB culture medium for bacterium solution of transferring
Element), 37 DEG C, 200rpm is cultivated 12 hours.
D. above-mentioned bacterium solution is suitably diluted, is coated on LAS solid medium tablets, 25 DEG C are cultivated 24 hours.
E. the single bacterium grown in LAS solid medium tablets in picking step d drops down onto 5mLLB culture medium, and 37 DEG C,
200rpm is cultivated 12 hours, is extracted genomic DNA, is used primer ldhA-1:ATCGGAATTCAGGGTATTGAGCTGGGCGTC
PCR amplification is carried out with the primer of primer ldhA-4:TCTACCCGGGTTTTCAGCCGCTTTCTCTCT composition, is verified through electrophoresis
To correct clone, the as Klebsiella of ldhA gene knockout.
LB culture medium: agar powder 15g/ is added in solid medium by peptone 10g/L, yeast powder 5g/L, NaCl 10g/L
L, 121 DEG C sterilize 20 minutes.
M9 solid medium: Na2HPO4·12H2O 1.7g/L、KH2PO40.3g/L、NaCl 0.05g/L、NH4Cl
0.1g/L, trisodium citrate 0.5g/L, agar powder 15g/L, 121 DEG C sterilize 20 minutes.
LAS solid medium: peptone 10g/L, yeast powder 5g/L, sucrose 150g/L, agar powder 15g/L, 115 DEG C go out
Bacterium 20 minutes.
Embodiment two: the knockout of Acetokinase gene ackA in Klebsiella
(1) clone of Acetokinase gene ackA upstream and downstream homeologous sequence
According to the method for 1 step of embodiment (1), design primer, the genomic DNA with Klebsiella ATCC 25955 is
Template, with primer ackA-1:ATCGGAATTCTAGCGGGTGGCACGAATAAT and primer ackA-2:
The primer of ATTAGGATCCGCTACCGCAGTTCAGAACCA composition obtains the upstream sequence of ackA gene after carrying out PCR amplification；
With primer ackA-3:GCAAGGATCCCTATACCATCGCACTGACCG and primer ackA-4:
The primer of TCCCCCCGGGCGAGACAAAAGACTTTCATC composition obtains the downstream sequence of ackA gene after carrying out PCR amplification.
PCR product is recycled and is purified, target fragment is obtained.
(2) suicide vectorBuilding
Using the homologous fragment of restriction enzyme EcoRI and BamHI digestion ackA upstream region of gene, using BamHI and
The homologous fragment of XmaI digestion ackA downstream of gene, using EcoRI and XmaI digestion pKR6K carrier, by digestion products and pKR6K
Carrier is recycled and is purified, using T4 ligase, by the homologous fragment of ackA upstream region of gene, downstream homologous fragment and
The connection of pKR6K carrier, connection product is transferred to Escherichia coli S17-1 (λ pir), is screened positive transformant, is obtained carrying ackA
The suicide vector of upstream region of gene and downstream part homologous fragment
(3) building for the Klebsiella that Acetokinase gene ackA is knocked out
It is carried what is obtained in step (2)Escherichia coli S17-1 (λ pir) (donor bacterium) and real
The Klebsiella (recipient bacterium) for applying the ldhA gene knockout obtained in example 1 carries out double parents, makesOn
AckA upstream region of gene and downstream homeologous sequence and Klebsiella genome occur homologous recombination, to make Cray primary
AckA gene delection inactivation in Salmonella, to obtain the Klebsiella of ldhA gene and ackA gene knockout.Specific method
It is same as Example 1, only PCR verify when, using primer ackA-1:ATCGGAATTCTAGCGGGTGGCACGAATAAT with draw
The primer of object ackA-4:TCCCCCCGGGCGAGACAAAAGACTTTCATC composition carries out PCR verifying.
Embodiment three: the knockout of acetonic acid oxidase gene poxB in Klebsiella
(1) clone of acetonic acid oxidase gene poxB upstream and downstream homeologous sequence
According to the method for 1 step of embodiment (1), design primer, the genomic DNA with Klebsiella ATCC 25955 is
Template, with primer poxB-1:GCATGAATTCTTTCGCTGCCACTTTATCCA and primer poxB-2:
The primer of ATTAGGATCCGGCGAAAACCAACTGGCTCA composition obtains the upstream sequence of poxB gene after carrying out PCR amplification；
With primer poxB-3:ATGCGGATCCACGGTCTGCTTCATGATCTC and primer poxB-4:
The primer of CGTACTGCAGATCTAAGCCGACCATCAGCC composition obtains the downstream sequence of poxB gene after carrying out PCR amplification.
PCR product is recycled and is purified, target fragment is obtained.
(2) suicide vectorBuilding
Using the homologous fragment of restriction enzyme EcoRI and BamHI digestion poxB upstream region of gene, using BamHI and
The homologous fragment of PstI digestion poxB downstream of gene, using EcoRI and PstI digestion pKR6K carrier, by digestion products and pKR6K
Carrier is recycled and is purified, using T4 ligase, by the homologous fragment of poxB upstream region of gene, downstream homologous fragment and
The connection of pKR6K carrier, connection product is transferred to Escherichia coli S17-1 (λ pir), is screened positive transformant, is obtained carrying poxB
The suicide vector of upstream region of gene and downstream part homologous fragment
(3) building for the Klebsiella that acetonic acid oxidase gene poxB is knocked out
It is carried what is obtained in step (2)Escherichia coli S17-1 (λ pir) (donor bacterium) and real
The Klebsiella (recipient bacterium) for applying the ldhA gene and ackA gene knockout that obtain in example 2 carries out double parents, makesOn poxB upstream region of gene and downstream homeologous sequence and Klebsiella genome occur it is homologous heavy
Group, to make the poxB gene delection in Klebsiella inactivate, to obtain ldhA gene, ackA gene and poxB clpp gene
The Klebsiella removed.Specific method is same as Example 1, when only PCR is verified, uses primer poxB-1:
GCATGAATTCTTTCGCTGCCACTTTATCCA and primer poxB-4:CGTACTGCAGATCTAAGCCGACCATCAGCC is formed
Primer carry out PCR verifying.
Example IV: the knockout of fumaric reductase gene frdA in Klebsiella
(1) clone of fumaric reductase gene frdA upstream and downstream homeologous sequence
According to the method for 1 step of embodiment (1), design primer, the genomic DNA with Klebsiella ATCC 25955 is
Template, with primer frdA-1:ATTCGAATTCGGTCTGCTTGTTTGGCCCCT and primer frdA-2:
The primer of CATGCATATGCCGTCTGGAATATGGCGATG composition obtains the upstream sequence of frdA gene after carrying out PCR amplification；
With primer frdA-3:ATTCCATATGCGTATGGCTGCGCATCGGAT and primer frdA-4:
The primer of TAACCTGCAGCGGTAAAGAAACGGCGGATT composition obtains the downstream sequence of frdA gene after carrying out PCR amplification.
PCR product is recycled and is purified, target fragment is obtained.
(2) suicide vectorBuilding
Using the homologous fragment of restriction enzyme EcoRI and NdeI digestion frdA upstream region of gene, NdeI and PstI are used
The homologous fragment of digestion frdA downstream of gene, using EcoRI and PstI digestion pKR6K carrier, by digestion products and pKR6K carrier
It is recycled and is purified, using T4 ligase, the homologous fragment of frdA upstream region of gene, the homologous fragment in downstream and pKR6K are carried
Body connection, connection product is transferred to Escherichia coli S17-1 (λ pir), is screened positive transformant, is obtained carrying on frdA gene
The suicide vector of trip and downstream part homologous fragment
(3) building for the Klebsiella that fumaric reductase gene frdA is knocked out
It is carried what is obtained in step (2)Escherichia coli S17-1 (λ pir) (donor bacterium) and real
Klebsiella (recipient bacterium) the progress parents' sheet for applying the ldhA gene obtained in example 3, ackA gene and poxB gene knockout is miscellaneous
It hands over, makesOn frdA upstream region of gene and downstream homeologous sequence and Klebsiella genome occur it is same
Source recombination, to make the frdA gene delection in Klebsiella inactivate, to obtain ldhA gene, ackA gene, poxB base
The Klebsiella of cause and frdA gene knockout.Specific method is same as Example 1, when only PCR is verified, uses primer
FrdA-1:ATTCGAATTCGGTCTGCTTGTTTGGCCCCT and primer frdA-4:
The primer of TAACCTGCAGCGGTAAAGAAACGGCGGATT composition carries out PCR verifying.
Embodiment five: the knockout of isocitrate lyase gene aceA in Klebsiella
(1) clone of isocitrate lyase gene aceA upstream and downstream homeologous sequence
According to the method for 1 step of embodiment (1), design primer, with the genomic DNA of Klebsiella ATCC 25955
For template, with primer aceA-1:ATGCGAATTCGAACGCACGGAAGCCGGAAT and primer aceA-2:
The primer of ATCGAAGCTTGCGGGTTCGTCCATTCTTTG composition obtains the upstream sequence of aceA gene after carrying out PCR amplification；
With primer aceA-3:ATCGAAGCTTTTGAGAAAGTGCAGCAGCCG and primer aceA-4:
The primer of ATGGCTGCAGAGGCCCACGTGGTGATCGTA composition obtains the downstream sequence of aceA gene after carrying out PCR amplification.
PCR product is recycled and is purified, target fragment is obtained.
(2) suicide vectorBuilding
Using the homologous fragment of restriction enzyme EcoRI and HindIII digestion aceA upstream region of gene, HindIII is used
With the homologous fragment of PstI digestion aceA downstream of gene, using EcoRI and PstI digestion pKR6K carrier, by digestion products and
PKR6K carrier is recycled and is purified, using T4 ligase, by the homologous fragment of aceA upstream region of gene, the homologous fragment in downstream
It is connected with pKR6K carrier, connection product is transferred to Escherichia coli S17-1 (λ pir), screened positive transformant, carried
The suicide vector of aceA upstream region of gene and downstream part homologous fragment
(3) building for the Klebsiella that isocitrate lyase gene aceA is knocked out
It is carried what is obtained in step (2)Escherichia coli S17-1 (λ pir) (donor bacterium) and real
The Klebsiella (recipient bacterium) for applying the ldhA gene obtained in example 4, ackA gene, poxB gene and frdA gene knockout carries out
Double parents, makeOn aceA upstream region of gene and downstream homeologous sequence and Klebsiella base
Because of a group generation homologous recombination, to make the aceA gene delection in Klebsiella inactivate, to obtain ldhA gene, ackA base
The Klebsiella engineering bacteria of cause, poxB gene, frdA gene and aceA gene knockout, is named as Klebsiella XP-5.Tool
Body method is same as Example 1, when only PCR is verified, uses primer aceA-1:
ATGCGAATTCGAACGCACGGAAGCCGGAAT and primer aceA-4:ATGGCTGCAGAGGCCCACGTGGTGATCGTA is formed
Primer carry out PCR verifying.
Embodiment six: the engineering bacteria XP-5 fermentation glycerol coproduction 1,3- the third two of lactic acid producing, acetic acid and succinate pathway missing
Alcohol, 2,3- butanediol and ethyl alcohol
(1) glycerol fermentation culture medium
Yeast powder 5g/L, K2HPO4·3H2O 10g/L、KH2PO42g/L、NH4Cl 1g/L、MgSO4·7H2O 0.1g/L、
FeCl3·6H2O 30mg/L、CoCl2·6H2O 5mg/L and glycerol 20g/L.121 DEG C of culture medium sterilize 20 minutes.
(2) seed culture
Klebsiella XP-5 is selected, the 500mL triangular flask equipped with glycerol fermentation culture medium is aseptically seeded to
In, it 30-37 DEG C, cultivates 12 hours in 100-200rpm shaking table, obtains seed culture fluid.
(3) fermented and cultured
Seed culture fluid obtained in step (2) is seeded in the 5L fermentor equipped with glycerol fermentation culture medium, is carried out
Fed batch fermentation, inoculum concentration are 0.5-5% (v/v), and the present embodiment is 1% (v/v), ventilatory capacity 0.5-2.0vvm, this reality
Applying example is 1.0vvm, speed of agitator 100-300rpm, the present embodiment 250rpm, and fermentation temperature is 30-37 DEG C, the present embodiment
It is 37 DEG C, in fermentation process, adjusting fermentation liquid pH using 2M NaOH solution is 6.0-7.0, the present embodiment 6.6.When glycerol is sent out
When 20g/L glycerol depletion in ferment culture medium, by adding 500-700g/L glycerite into fermentor, the present embodiment be to
The glycerite of 700g/L is added in fermentor, controlling glycerol concentration in fermentation liquid is 5-30g/L.When 1,3- third in fermentation liquid
When the concentration of glycol, 2,3-butanediol and ethyl alcohol no longer increases, stop fermentation.
(4) fermentation results
Using obtained in embodiment 5 lactic acid producing, acetic acid and succinate pathway missing engineering bacteria XP-5 as experimental group, with gram
The primary Salmonella wild mushroom of thunder is control group, carries out fed batch fermentation.The fermentation of engineering bacteria XP-5 carries out 34 hours, and fermentation results are such as
Shown in Fig. 2 and table 1.Lactic acid producing, the engineering bacteria XP-5 vitro growth rates of acetic acid and succinate pathway missing and glycerol metabolism speed
Degree slows down compared with wild mushroom, but the yield of organic acid (lactic acid, acetic acid and succinic acid) reduces 85.4% compared with wild mushroom；Meanwhile
The output increased of total alcohol (1,3- propylene glycol, 2,3- butanediol and ethyl alcohol) 31.7%.Wherein, the yield of 1,3-PD from
64.3g/L is improved to 74.5g/L, improves 15.9%；The yield of 2,3-butanediol is improved from 10.2g/L to 20.8g/L, is improved
104.9%；The yield of ethyl alcohol is improved from 5.2g/L to 9.7g/L, improves 86.5%；Total conversion of three kinds of alcohol to glycerol
Reach 0.72g/g, improve 41.2%, realizes the coproduction of 1,3-PD, 2,3-butanediol and ethyl alcohol.
Table 1: the product of Klebsiella engineering bacteria XP-5 and wild mushroom fermentation glycerol comparison
Claims (5)
1. one plant produces the engineering bacteria that organic acid approach lacks, which is characterized in that the lactic acid producing way of Klebsiella wild-type strain
Diameter produces acetate pathway and produces succinate pathway missing, which is named as Klebsiella XP-5, on March 6th, 2015
It is preserved in China typical culture collection center, deposit number are as follows: CCTCC M 2015092.
2. the engineering bacteria according to claim 1 for producing organic acid approach missing, which is characterized in that the lactic acid producing approach lacks
Mistake is inactivation lactic acid dehydrogenase gene；The production acetate pathway missing is inactivation Acetokinase gene and pyruvate oxidase base
Cause；The production succinate pathway missing is inactivation fumaric reductase gene and isocitrate lyase gene.
3. the engineering bacteria according to claim 2 for producing organic acid approach missing, which is characterized in that the lactic dehydrogenase base
Because of sequence such as SEQ ID NO.1, the Acetokinase gene sequence such as SEQ ID NO.2, the acetonic acid oxidase gene sequence
It arranges such as SEQ ID NO.3, the fumaric reductase gene order such as SEQ ID NO.4, the isocitrate lyase gene
Sequence such as SEQ ID NO.5.
4. the engineering bacteria for producing organic acid approach missing of claim 1 is in coproduction 1,3- propylene glycol, 2,3- butanediol and ethyl alcohol
Application method, its step are as follows:
(1) seed culture: Klebsiella XP-5CCTCC M 2015092 is selected, is aseptically seeded to equipped with glycerol
In the 500mL triangular flask of fermentation medium, 30-37 DEG C, cultivates 8-15 hours in 100-200rpm shaking table, obtain seed culture
Liquid；
(2) seed culture fluid obtained in step (1) fermented and cultured: is seeded to the 5L fermentor equipped with glycerol fermentation culture medium
In, fed batch fermentation is carried out, inoculum concentration is 0.5-5% (v/v), ventilatory capacity 0.5-2.0vvm, speed of agitator 100-
300rpm, fermentation temperature are 30-37 DEG C, and in fermentation process, adjusting fermentation liquid pH using aqueous slkali is 6.0-7.0, when glycerol is sent out
When glycerol depletion in ferment culture medium, by adding the glycerite of 500-700g/L into fermentor, control sweet in fermentation liquid
Oil concentration is 5-30g/L, when the concentration of 1,3-PD, 2,3-butanediol and ethyl alcohol in fermentation liquid no longer increases, stops hair
Ferment.
5. the engineering bacteria according to claim 4 for producing organic acid approach missing is in coproduction 1,3- propylene glycol, 2,3- butanediol
With the application method in ethyl alcohol, which is characterized in that contain glycerol as carbon source in the glycerol fermentation culture medium.
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