CN107325999A - Klebsiella for enhancing expression of citT gene and application of Klebsiella for producing 1, 3-propylene glycol - Google Patents
Klebsiella for enhancing expression of citT gene and application of Klebsiella for producing 1, 3-propylene glycol Download PDFInfo
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- CN107325999A CN107325999A CN201710680961.5A CN201710680961A CN107325999A CN 107325999 A CN107325999 A CN 107325999A CN 201710680961 A CN201710680961 A CN 201710680961A CN 107325999 A CN107325999 A CN 107325999A
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- 101150103610 citT gene Proteins 0.000 title claims abstract description 131
- 241000588748 Klebsiella Species 0.000 title claims abstract description 111
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 230000014509 gene expression Effects 0.000 title claims abstract description 19
- 230000002708 enhancing effect Effects 0.000 title abstract 4
- 238000000855 fermentation Methods 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 50
- 230000002018 overexpression Effects 0.000 claims description 83
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 82
- 238000011218 seed culture Methods 0.000 claims description 42
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 41
- 239000013612 plasmid Substances 0.000 claims description 38
- 235000011187 glycerol Nutrition 0.000 claims description 33
- 238000004519 manufacturing process Methods 0.000 claims description 33
- 229920000166 polytrimethylene carbonate Polymers 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 24
- 239000002253 acid Substances 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 19
- 239000013604 expression vector Substances 0.000 claims description 18
- 239000012531 culture fluid Substances 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 239000012530 fluid Substances 0.000 claims description 13
- 229960005091 chloramphenicol Drugs 0.000 claims description 11
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 11
- 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 claims description 9
- 229930027917 kanamycin Natural products 0.000 claims description 9
- 241000607142 Salmonella Species 0.000 claims description 7
- 206010035664 Pneumonia Diseases 0.000 claims description 2
- -1 Propyl dithiocarbamate galactoside Chemical class 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 description 20
- 235000013619 trace mineral Nutrition 0.000 description 15
- 239000011573 trace mineral Substances 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 10
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 10
- 239000000126 substance Substances 0.000 description 9
- 238000010276 construction Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- 239000007836 KH2PO4 Substances 0.000 description 6
- 241000588747 Klebsiella pneumoniae Species 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 6
- 229910052564 epsomite Inorganic materials 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 239000001509 sodium citrate Substances 0.000 description 6
- 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 6
- 229940038773 trisodium citrate Drugs 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 5
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 5
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 5
- 239000006137 Luria-Bertani broth Substances 0.000 description 5
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 5
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 5
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 5
- 210000004209 hair Anatomy 0.000 description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 5
- 239000011565 manganese chloride Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000011684 sodium molybdate Substances 0.000 description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- 108010044467 Isoenzymes Proteins 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 239000005515 coenzyme Substances 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 238000005728 strengthening Methods 0.000 description 4
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 3
- 229940035437 1,3-propanediol Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241001014264 Klebsiella variicola Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 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
- 238000012407 engineering method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229920002215 polytrimethylene terephthalate Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001172 regenerating effect Effects 0.000 description 3
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 3
- 101100098786 Bacillus subtilis (strain 168) tapA gene Proteins 0.000 description 2
- 101100321116 Escherichia coli (strain K12) yqhD gene Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108010025885 Glycerol dehydratase Proteins 0.000 description 2
- 241000588749 Klebsiella oxytoca Species 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 101150086278 fdh gene Proteins 0.000 description 2
- 238000012215 gene cloning Methods 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010011958 1,3-propanediol dehydrogenase Proteins 0.000 description 1
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 description 1
- BRARRAHGNDUELT-UHFFFAOYSA-N 3-hydroxypicolinic acid Chemical compound OC(=O)C1=NC=CC=C1O BRARRAHGNDUELT-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000005314 correlation function Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 101150061843 dhaT gene Proteins 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- RZTAMFZIAATZDJ-UHFFFAOYSA-N felodipine Chemical compound CCOC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC(Cl)=C1Cl RZTAMFZIAATZDJ-UHFFFAOYSA-N 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229940090013 plendil Drugs 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical class CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 101150113187 yqhD gene Proteins 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/26—Klebsiella (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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- General Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides Klebsiella for enhancing expression of citT gene and application thereof in producing 1, 3-propylene glycol. The Klebsiella for enhancing the expression of the citT gene is obtained by enhancing the expression of the citT gene in the Klebsiella. The Klebsiella with the enhanced expression of the citT gene is used for producing the 1, 3-propylene glycol by fermentation, and the yield of the 1, 3-propylene glycol can be improved.
Description
Technical field
The present invention is the application of the Klebsiella and its production 1,3- propane diols on a kind of overexpression citT genes.
Background technology
1,3-PD is widely used, is very important chemical products and platform chemicals, can be used as adhesive, protection
Agent, lubricant, antifreeze and solvent, all have extensive use in high polymer material, medicine, chemical industry, food and cosmetic field.
Its most important purposes is production polytrimethylene terephthalate (PTT).PTT superperformance, including washing property, ductility and
Recovery all causes its market demand to expand day by day.Thus pulled 1,3- propane diols the market demand (Liu H.et al.,
Biotechnol J.2010,5(11):1137–1148)。
In view of the important function of 1,3-PD, a variety of drawbacks of chemical method synthesis, utilize microbial fermentation glycerol production
The research of 1,3- propane diols receives much concern.The bacterial strain for the synthesis 1,3- propane diols reported at present mainly has Klebsiella
(Klebsiella), fusobacterium, Enterobacter and genus lactubacillus.Wherein, the bacterial strain of Klebsiella belongs to amphimicrobian
Bacterium, easily cultivates in commercial Application, and such bacterial strain has good substrate glycerol tolerance and product 1,3-PD resistance to
By property, the production intensity and conversion ratio of 1,3-PD are higher.Therefore, Klebsiella is more excellent 1,3- of research
Propane diols production bacterial strain (Celi ń ska E.Crit Rev Biotechnol.2012,32 (3):274–288).Further to carry
The high 1,3-PD level of production, reduces 1,3-PD production cost, it is necessary to be transformed Klebsiella and optimized, with
The performances such as yield, conversion ratio and the production intensity of bacterial strain production 1,3-PD are improved, building genetic engineering Klebsiella is
Realize one of effective ways of above-mentioned target.At present, existing a variety of methods are used for the gene work for building production 1,3-PD
Journey Klebsiella：
(1) by key enzyme in gene engineering method overexpression 1,3-PD route of synthesis, such as glycerol dehydratase and
1,3- methyl glycol oxidoreductases (or its isoenzymes) (Zheng, P., Wereath, K., Sun, J., van den Heuvel,
J.,&Zeng,A.P.(2006).Overexpression of genes of the dha regulon and its
effects on cell growth,glycerol fermentation to 1,3-propanediol and plasmid
stability in Klebsiella pneumoniae.Process Biochemistry,41(10),2160-2169；Zhu,
J.G.,Li,S.,Ji,X.J.,Huang,H.,&Hu,N.(2009).Enhanced 1,3-propanediol production
in recombinant Klebsiella pneumoniae carrying the gene yqhD encoding 1,3-
propanediol oxidoreductase isoenzyme.World Journal of Microbiology and
Biotechnology,25(7),1217.).The yqhD gene clonings of Escherichia coli are inserted anti-with tetracycline by Zhu et al.
On the pUC18-tet plasmids of property gene, the overexpression plasmid pUC18-yqhD-tet of yqhD genes is obtained.By plasmid
PUC18-yqhD-tet is converted into Friedlander's bacillus, builds obtained engineering strain production 1,3-PD concentration
It is the 125.33% of control strain, the conversion ratio of engineering strain 1,3-PD is the 121.56% of control strain.Reinforcing
It is the effective of the raising 1,3- propane diols levels of production that expression 1,3- methyl glycol oxidoreductases isoenzymes, which builds engineering strain,
Method.Overexpression glycerol dehydratase and dhaT (or its isoenzymes) can increase the amount of these enzymes,
So as to improve the yield and production intensity of 1,3- propane diols.
(2) encoding gene (CN200510127744.0 of the accessory substance of 1,3- propane diols synthesis is knocked out；
CN201310071754.1).The encoding gene of the accessory substance of 1,3-PD synthesis is knocked out, accessory substance synthesis way can be blocked
Footpath, reduces accessory substance generation, improves 1,3-PD conversion ratio.
(3) built in the Klebsiella of production 1,3- propane diols coenzyme NAD H regenerative systems (Zhang, Y., Huang,
Z.,Du,C.,Li,Y.,&Cao,Z.A.(2009).Introduction of an NADH regeneration system
into Klebsiella oxytoca leads to an enhanced oxidative and reductive
metabolism of glycerol.Metabolic Engineering,11(2),101-106.).By Plendil Candida
Fdh gene clonings, and insert on pMAL-p2X plasmids, obtain the overexpression plasmid pMAL-p2X-fdh of fdh genes.Will
Plasmid pMAL-p2X-fdh is converted into acid-producing Klebsiella bacterium, is built obtained engineering strain and is produced 1,3-PD
Conversion ratio is the 117.3% of control strain.It is to improve the life of 1,3- propane diols to introduce regenerating coenzyme system constructing engineering strain
The effective ways of production level.More coenzyme NAD H can be provided for 1,3-PD synthesis by building coenzyme NAD H regenerative systems, be carried
High 1,3- propanediols.
(4) resistance is improved using gene engineering method transformation 1,3- propane diols production bacterial strains
(CN200810114647.1).The poisonous intermediate metabolites that bacterial strain is synthesized to 1,3- propane diols can be improved by improving bacterial strain resistance
The resistance of 3-HPA, reduces the anomaly in fermentation process.
(5) using gene engineering method transformation 1,3- propane diols production bacterial strain tricarboxylic acid cycle relevant enzyme expression quantity
(CN201110069870.0).The expression quantity of tricarboxylic acid cycle relevant enzyme malate dehydrogenase is improved, accelerates tricarboxylic acid cycle, is improved
ATP and NADH growing amount, improves 1,3-PD production intensity and conversion ratio.
The genome annotation result of Klebsiella shows that many Klebsiellas all have citT genes, still, at present
Research also not to the correlation function of Klebsiella citT genes is reported.
The content of the invention
Inventor has found that overexpression citT genes, are remarkably improved Cray in Klebsiella under study for action
Primary Salmonella produces the yield of 1,3- propane diols.
On the one hand, the invention provides a kind of Klebsiella of overexpression citT genes.
The Klebsiella of the overexpression citT genes of the present invention, it is by the overexpression in Klebsiella
Obtained from citT genes.
According to specific embodiments of the present invention, the Klebsiella of overexpression citT genes of the invention, it is logical
Cross in Friedlander's bacillus, acid-producing Klebsiella bacterium or become in Klebsiella of dwelling obtained from overexpression citT genes.
The Klebsiella of the overexpression citT genes of the present invention, wherein, the citT genes are from citric acid
The citT genes of bacterium.
According to specific embodiments of the present invention, the Klebsiella of overexpression citT genes of the invention, wherein, institute
It is from Friedlander's bacillus, acid-producing Klebsiella bacterium or the citT genes for becoming Klebsiella of dwelling to state citT genes.
According to specific embodiments of the present invention, the Klebsiella of overexpression citT genes of the invention, the lung
Scorching Klebsiella is Friedlander's bacillus ATCC 25955, and the acid-producing Klebsiella bacterium is acid-producing Klebsiella bacterium ATCC
BAA-1236, change Klebsiella of dwelling is dwelt Klebsiella ATCC BAA-830 into change.
In the present invention, Klebsiella (Klebsiella variicola) ATCC BAA-830 are dwelt in the change can be from the U.S.
Type culture deposit storehouse (American type culture collection, ATCC) purchase is obtained, its genome sequence
Reference can be made to NCBI record (preserving number of ncbi database is NZ_CP010523.2)；Friedlander's bacillus (Klebsiella
Pneumoniae) ATCC 25955 can deposit storehouse (American type culture from American Type Culture
Collection, ATCC) purchase obtains, and its genome sequence can be found in NCBI record, and (preserving number of ncbi database is
AQQH00000000)；Acid-producing Klebsiella bacterium (Klebsiella oxytoca) ATCC BAA-1236 can be from US mode culture
Thing deposit storehouse (American type culture collection, ATCC) purchase is obtained.
On the other hand, present invention also offers the method for the Klebsiella of the overexpression citT genes described in structure.
According to specific embodiments of the present invention, the technology of overexpression citT genes can be using in Klebsiella
Any feasible technology known.
According to the preferred embodiment of the present invention, the Cray of the overexpression citT genes described in structure of the invention
The method of primary Salmonella includes：
CitT genes are connected to expression vector, structure obtains citT overexpression plasmids；
CitT overexpression plasmids are transformed into Klebsiella, on the resistance culture base of kanamycins or chloramphenicol
Culture obtains positive colony, the Klebsiella for the expression citT genes that strengthened.
According to the preferred embodiment of the present invention, the Cray of the overexpression citT genes described in structure of the invention
In the method for primary Salmonella, the expression vector includes pDK6 or pDK7.
In some specific embodiments of the present invention, the Cray of the overexpression citT genes described in structure of the invention
The method of primary Salmonella includes：
Step one, using the DNA of the genome sequence of Klebsiella as template, primer is designed, by PCR by its citT base
Because of complete clone, citT genes are obtained；
Step 2, expression vector is connected to by citT genes, and structure obtains citT overexpression plasmids；
Step 3, citT overexpression plasmids are transformed into host's Klebsiella, in kanamycins or chloramphenicol
Culture obtains the Klebsiella of positive colony, as overexpression citT genes on resistance culture base.
In the construction method of the Klebsiella of above-mentioned overexpression citT genes, it is preferable that in step one, the Cray
Primary Salmonella be change dwell Klebsiella ATCC BAA-830 when, design of primers is：
Kv-citT-F：GGCCGAATTCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 1)
Kv-citT-R：GAGCGGATCCTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 2).
Note：“Kv-citT-F”：Refer to using change dwell Klebsiella ATCC BAA-830 when, expand citT genes forward direction
Primer；" Kv-citT-R " refer to using change dwell Klebsiella ATCC BAA-830 when, expand citT genes reverse primer；Under
State the like, repeat no more.
In the construction method of the Klebsiella of above-mentioned overexpression citT genes, it is preferable that in step one, the Cray
When primary Salmonella is Friedlander's bacillus ATCC 25955, design of primers is：
Kp-citT-F：GAGCGGATCCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 3)
Kp-citT-R：GGCCAAGCTTTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 4).
In the construction method of the Klebsiella of above-mentioned overexpression citT genes, it is preferable that in step 2, citT is built
The method of overexpression plasmid is：
When expression vector is pDK6, using EcoRI and BamHI digestion citT genes and expression vector pDK6, by citT
Gene is connected between expression vector pDK6 EcoRI and BamHI restriction enzyme sites, builds obtained citT overexpression plasmids
pDK6-citT；
, will using BamHI and HindIII digestion citT genes and expression vector pDK7 when expression vector is pDK7
CitT genes are connected between expression vector pDK7 BamHI and HindIII restriction enzyme sites, build obtained citT overexpression matter
Grain pDK7-citT.
In the construction method of the Klebsiella of above-mentioned overexpression citT genes, it is preferable that the citT is strengthened into table
After being transformed into up to plasmid in host's Klebsiella, the cell after conversion is coated on containing 20-50 μ g/mL kanamycins or chlorine
On the solid LB media flat board of mycin, in 37 DEG C of quiescent cultures 24 hours, positive colony is obtained, is overexpression citT
The Klebsiella of gene.
On the other hand, present invention also offers the Klebsiella of described overexpression citT genes in production 1,3- third
Application in glycol.
The opposing party, present invention also offers a kind of method for producing 1,3-PD, this method includes：
The Klebsiella of overexpression citT genes of the present invention is inoculated in seed culture medium, Shaking culture
Obtain seed culture fluid；
Seed culture fluid is inoculated in the fermentation medium containing glycerine and cultivated, production obtains 1,3-PD.
According to specific embodiments of the present invention, in the method for production 1,3-PD of the invention, the bar of Shaking culture
Part is 32-37 DEG C, rotating speed is that 150-200rpm, the pH of seed culture medium are 6.8-7.0, cultivates 10-12h.More specifically, described
In seed culture medium, in terms of 1L seed culture mediums, the seed culture medium includes 10g peptone, 5g dusty yeast, 10g chlorine
Change sodium and 50mg kanamycins；Or, the peptone of the seed culture medium including 10g, 5g dusty yeast, 10g sodium chloride and
20-25mg chloramphenicol.Preferably, the Klebsiella of access overexpression citT genes in every 100mL seed culture mediums
Inclined-plane lawn 1-5 rings.
According to specific embodiments of the present invention, in the method for production 1,3-PD of the invention, the bar of fermented and cultured
Part is 32-37 DEG C, rotating speed is that 100-150rpm, throughput are that 0.5-1.0vvm, fermentation medium pH value are 6.5-6.8, fermentation
30-35h.Preferably, glycerol adding is flowed in fermentation process makes glycerol concentration in zymotic fluid control in 10-60g/L.More specifically, institute
The volume ratio for stating seed culture fluid and the fermentation medium containing glycerine is (0.1-1):100.
According to specific embodiments of the present invention, in the method for production 1,3-PD of the invention, the fermented and cultured
Base, in terms of 1L fermentation mediums, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g KH2PO4, 1g
NH4Cl, 0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 50mg kanamycins and 0.3mL micro member
Plain solution.Or, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g KH2PO4, 1g NH4Cl、
0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 20mg chloramphenicol and 0.3mL trace element solution.
More specifically, wherein, in terms of 1L trace element solutions, the trace element solution includes 7.56g ZnCl2, 15.65g
FeCl3·6H2O, 6.28g MnCl2·4H2O, 1.32g CuCl2·2H2O, 6.42g CoCl2·6H2O, 0.48g's
H3BO3With 3.22g Na2MoO4·2H2O。
According to specific embodiments of the present invention, in the method for production 1,3-PD of the invention, in fermentation 3-4h
When, the isopropylthiogalactoside (IPTG) that 0.01-1mM is added into zymotic fluid induces the overexpression of citT genes.
The engineering strain built using the inventive method, by strengthening the expression of citT genes, can improve 1,
The yield of ammediol.The some embodiments of the present invention show, carry plasmid pDK6-citT and strengthen citT gene expressions
Friedlander's bacillus (Klebsiella pneumoniae) ATCC 25955 1,3- propanediols be not carry plasmid
The 125% of pDK6-citT wild strain；Carry plasmid pDK7-citT and strengthen the e coil k 1 pneumonia of citT gene expressions
Bacterium (Klebsiella pneumoniae) ATCC 25955 1,3- propanediols are the open countries for not carrying plasmid pDK7-citT
The 118% of raw bacterial strain；Carry plasmid pDK7-citT and strengthen the acid-producing Klebsiella bacterium (Klebsiella of citT gene expressions
Oxytoca) ATCC BAA-1236 1,3- propanediols are the 131% of the wild strain for not carrying plasmid pDK7-citT；
The change for carrying plasmid pDK7-citT and strengthening citT expression is dwelt Klebsiella (Klebsiella variicola) ATCC
BAA-830 1,3- propanediols are the 129% of the wild strain for not carrying plasmid pDK7-citT.
Embodiment
In order to which technical characteristic, purpose and beneficial effect to the present invention are more clearly understood from, now to the skill of the present invention
Art scheme carry out it is described further below, but it is not intended that to the present invention can practical range restriction.
The reagent that PCR is used in embodiment is conventional commercial product, routine of the application method with reference to product description
Method.
Detect that 1,3- content of propylene glycol is detected by gas chromatograph in zymotic fluid in embodiment.Wherein, gas phase
Chromatograph model GC-2014C, purchased from Japanese Shimadzu Corporation, chromatographic column uses capillary column AT.SE-54 (30m × 0.53mm
× 1.0 μm), fid detector, N2As carrier gas, column temperature is 120 DEG C, and detector is 250 DEG C with injector temperature.
Embodiment 1
The present embodiment provides a kind of construction method of overexpression citT genetic engineering Friedlander's bacillus, it include with
Lower step：
Step one, using Klebsiella ATCC BAA-830 genome sequence, (preserving number of ncbi database is NZ_
CP010523.2 DNA) is template, designs primer, and design of primers is：
Kv-citT-F：GGCCGAATTCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 1)
Kv-citT-R：GAGCGGATCCTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 2),
Its citT gene is completely cloned by PCR, citT genes are obtained.
Step 2, using EcoRI and BamHI digestion citT genes and expression vector pDK6, table is connected to by citT genes
Up between carrier pDK6 EcoRI and BamHI restriction enzyme sites, structure obtains citT overexpression plasmids pDK6-citT.
Step 3, prepares electric transformed competence colibacillus Friedlander's bacillus ATCC 25955, by citT overexpression plasmids
PDK6-citT is transformed into Friedlander's bacillus ATCC 25955, by the cell after conversion be coated on containing 50 μ g/mL cards that
On the solid LB media flat board of mycin, in 37 DEG C of quiescent cultures 24 hours, positive colony is obtained, is overexpression citT
Genetic engineering Friedlander's bacillus.
The present embodiment also provides overexpression citT genetic engineerings Friedlander's bacillus or Friedlander's bacillus
The method that ATCC 25955 produces 1,3-PD, comprises the following steps：
Step (1), by overexpression citT genetic engineerings Friedlander's bacillus or Friedlander's bacillus ATCC
The ring of 25955 inclined-plane lawn 1 is inoculated in seed culture medium, and Shaking culture obtains seed culture fluid；Wherein, with 1L seed culture mediums
Meter, the seed culture medium includes 10g peptone, 5g dusty yeast, 10g sodium chloride and 50mg kanamycins；Shaking flask is trained
Foster condition is 37 DEG C, rotating speed is that 200rpm, the pH of seed culture medium are 7.0, cultivates 12h.
In step (2), the fermentation tank that seed culture fluid is inoculated in the fermentation medium containing glycerine, culture obtains 1,3-
Propane diols；The volume ratio of the seed culture fluid and the fermentation medium containing glycerine is 1：100.
Wherein, in terms of 1L fermentation mediums, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g's
KH2PO4, 1g NH4Cl, 0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 50mg kanamycins and
0.3mL trace element solution, in terms of 1L trace element solutions, the trace element solution includes 7.56g ZnCl2、15.65g
FeCl3·6H2O, 6.28g MnCl2·4H2O, 1.32g CuCl2·2H2O, 6.42g CoCl2·6H2O, 0.48g's
H3BO3With 3.22g Na2MoO4·2H2O；The condition of fermented and cultured is 37 DEG C, rotating speed is that 150rpm, throughput are 1.0vvm, hair
Ferment Medium's PH Value is 6.5, and ferment 30h；
In the 3h of fermentation, 0.1mM isopropylthiogalactoside (IPTG) induction citT is added into fermentation tank
The overexpression of gene, and stream glycerol adding makes glycerol concentration in zymotic fluid control in 10-20g/L during the fermentation.
Using the concentration of 1,3-PD in gas chromatographic detection zymotic fluid, Friedlander's bacillus ATCC 25955 ferments
The concentration for producing 1,3-PD is 60g/L, and overexpression is from the citT genes for becoming Klebsiella ATCC BAA-830 of dwelling
Genetic engineering Friedlander's bacillus production 1,3-PD concentration be 75g/L, yield is Friedlander's bacillus ATCC
The 125% of 25955 yield.
Embodiment 2
The present embodiment provides a kind of construction method of overexpression citT genetic engineering Friedlander's bacillus, it include with
Lower step：
Step one, using Friedlander's bacillus ATCC 25955 genome sequence (preserving number of ncbi database as
AQQH00000000 DNA) is template, designs primer, and design of primers is：
Kp-citT-F：GAGCGGATCCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 3)
Kp-citT-R：GGCCAAGCTTTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 4),
Its citT gene is completely cloned by PCR, citT genes are obtained.Pass through detection, Friedlander's bacillus ATCC
The nucleotide sequence of citT gene of the 25955 citT genes with becoming Klebsiella ATCC BAA-830 of dwelling is consistent with 93%
Property, amino acid sequence has 97% uniformity.
Step 2, using BamHI and HindIII digestion citT genes and expression vector pDK7, citT genes are connected to
Between expression vector pDK7 BamHI and HindIII restriction enzyme sites, structure obtains citT overexpression plasmids pDK7-citT.
Step 3, prepares electric transformed competence colibacillus Friedlander's bacillus ATCC 25955, by citT overexpression plasmids
PDK7-citT is transformed into Friedlander's bacillus ATCC 25955, the cell after conversion is coated on mould containing 20 μ g/mL chlorine
On the solid LB media flat board of element, in 37 DEG C of quiescent cultures 24 hours, positive colony is obtained, is overexpression citT bases
Because of engineering Friedlander's bacillus.
The present embodiment also provides overexpression citT genetic engineerings Friedlander's bacillus or Friedlander's bacillus
The method that ATCC 25955 produces 1,3-PD, comprises the following steps：
Step (1), by overexpression citT genetic engineerings Friedlander's bacillus or Friedlander's bacillus ATCC
The ring of 25955 inclined-plane lawn 3 is inoculated in seed culture medium, and Shaking culture obtains seed culture fluid；Wherein, with 1L seed culture mediums
Meter, the seed culture medium includes 10g peptone, 5g dusty yeast, 10g sodium chloride and 20mg chloramphenicol；Shaking culture
Condition be 37 DEG C, rotating speed be that 150rpm, the pH of seed culture medium are 6.8, cultivate 10h.
In step (2), the fermentation tank that seed culture fluid is inoculated in the fermentation medium containing glycerine, culture obtains 1,3-
Propane diols；The volume ratio of the seed culture fluid and the fermentation medium containing glycerine is 1：100.
Wherein, in terms of 1L fermentation mediums, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g's
KH2PO4, 1g NH4Cl, 0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 20mg chloramphenicol and 0.3mL
Trace element solution, in terms of 1L trace element solutions, the trace element solution includes 7.56g ZnCl2, 15.65g
FeCl3·6H2O, 6.28g MnCl2·4H2O, 1.32g CuCl2·2H2O, 6.42g CoCl2·6H2O, 0.48g's
H3BO3With 3.22g Na2MoO4·2H2O；The condition of fermented and cultured is 37 DEG C, rotating speed is that 100rpm, throughput are 0.8vvm, hair
Ferment Medium's PH Value is 6.8, and ferment 35h；
In the 3h of fermentation, 0.1mM isopropylthiogalactoside (IPTG) induction citT is added into fermentation tank
The overexpression of gene, and stream glycerol adding makes glycerol concentration in zymotic fluid control in 10-20g/L during the fermentation.
Using the concentration of 1,3-PD in gas chromatographic detection zymotic fluid, Friedlander's bacillus ATCC 25955 ferments
The concentration for producing 1,3-PD is 60g/L, and the genetic engineering Friedlander's bacillus of its own citT gene of overexpression is given birth to
The concentration for producing 1,3-PD is 71g/L, and yield is the 118% of the yield of Friedlander's bacillus ATCC 25955.
Embodiment 3
The present embodiment provides a kind of construction method of overexpression citT genetic engineering acid-producing Klebsiella bacteriums, it include with
Lower step：
Step one, using Friedlander's bacillus ATCC 25955 genome sequence (preserving number of ncbi database as
AQQH00000000 DNA) is template, designs primer, and design of primers is：
Kp-citT-F：GAGCGGATCCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 3)
Kp-citT-R：GGCCAAGCTTTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 4),
Its citT gene is completely cloned by PCR, citT genes are obtained.Pass through detection, Friedlander's bacillus ATCC
The nucleotide sequence of citT gene of the 25955 citT genes with becoming Klebsiella ATCC BAA-830 of dwelling is consistent with 93%
Property, amino acid sequence has 97% uniformity.
Step 2, using BamHI and HindIII digestion citT genes and expression vector pDK7, citT genes are connected to
Between expression vector pDK7 BamHI and HindIII restriction enzyme sites, structure obtains citT overexpression plasmids pDK7-citT.
Step 3, prepares electric transformed competence colibacillus acid-producing Klebsiella bacterium ATCC BAA-1236, by citT overexpression plasmids
PDK7-citT is transformed into acid-producing Klebsiella bacterium ATCC BAA-1236, and the cell after conversion is coated on containing 20 μ g/mL
On the solid LB media flat board of chloramphenicol, in 37 DEG C of quiescent cultures 24 hours, positive colony is obtained, is the overexpression
CitT genetic engineering acid-producing Klebsiella bacteriums.
The present embodiment also provides overexpression citT genetic engineerings acid-producing Klebsiella bacterium or acid-producing Klebsiella bacterium
The method that ATCC BAA-1236 produce 1,3-PD, comprises the following steps：
Step (1), by overexpression citT genetic engineerings acid-producing Klebsiella bacterium or acid-producing Klebsiella bacterium ATCC BAA-
The ring of 1236 inclined-plane lawn 3 is inoculated in seed culture medium, and Shaking culture obtains seed culture fluid；Wherein, with 1L seed culture mediums
Meter, the seed culture medium includes 10g peptone, 5g dusty yeast, 10g sodium chloride and 20mg chloramphenicol；Shaking culture
Condition be 37 DEG C, rotating speed be that 150rpm, the pH of seed culture medium are 6.8, cultivate 10h.
In step (2), the fermentation tank that seed culture fluid is inoculated in the fermentation medium containing glycerine, culture obtains 1,3-
Propane diols；The volume ratio of the seed culture fluid and the fermentation medium containing glycerine is 1：100.
Wherein, in terms of 1L fermentation mediums, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g's
KH2PO4, 1g NH4Cl, 0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 20mg chloramphenicol and 0.3mL
Trace element solution, in terms of 1L trace element solutions, the trace element solution includes 7.56g ZnCl2, 15.65g
FeCl3·6H2O, 6.28g MnCl2·4H2O, 1.32g CuCl2·2H2O, 6.42g CoCl2·6H2O, 0.48g's
H3BO3With 3.22g Na2MoO4·2H2O；The condition of fermented and cultured is 37 DEG C, rotating speed is that 100rpm, throughput are 0.8vvm, hair
Ferment Medium's PH Value is 6.8, and ferment 35h；
In the 3h of fermentation, 0.01mM isopropylthiogalactoside (IPTG) induction citT is added into fermentation tank
The overexpression of gene, and stream glycerol adding makes glycerol concentration in zymotic fluid control in 20-40g/L during the fermentation.
Use the concentration of 1,3-PD in gas chromatographic detection zymotic fluid, acid-producing Klebsiella bacterium ATCC BAA-1236
The concentration of producing 1,3-propanediol through fermentation is 55g/L, citT base of the overexpression from Friedlander's bacillus ATCC 25955
The concentration of the genetic engineering acid-producing Klebsiella bacterium production 1,3-PD of cause is 72g/L, and yield is acid-producing Klebsiella bacterium
The 131% of ATCC BAA-1236 yield.
Embodiment 4
The present embodiment provides the construction method that a kind of overexpression citT genetic engineerings become Klebsiella of dwelling, it include with
Lower step：
Step one, using Friedlander's bacillus ATCC 25955 genome sequence (preserving number of ncbi database as
AQQH00000000 DNA) is template, designs primer, and design of primers is：
Kp-citT-F：GAGCGGATCCATGAAAGAGAAAAAGACAAC (such as SEQ ID NO：Shown in 3)
Kp-citT-R：GGCCAAGCTTTCAGAACATCATGGACATCC (such as SEQ ID NO：Shown in 4),
Its citT gene is completely cloned by PCR, citT genes are obtained.Pass through detection, Friedlander's bacillus ATCC
The nucleotide sequence of citT gene of the 25955 citT genes with becoming Klebsiella ATCC BAA-830 of dwelling is consistent with 93%
Property, amino acid sequence has 97% uniformity.
Step 2, using BamHI and HindIII digestion citT genes and expression vector pDK7, citT genes are connected to
Between expression vector pDK7 BamHI and HindIII restriction enzyme sites, structure obtains citT overexpression plasmids pDK7-citT.
Step 3, prepares electric transformed competence colibacillus and becomes Klebsiella ATCC BAA-830 of dwelling, by citT overexpression plasmids
PDK7-citT is transformed into change and dwelt in Klebsiella ATCC BAA-830, and the cell after conversion is coated on containing 25 μ g/mL chlorine
On the solid LB media flat board of mycin, in 37 DEG C of quiescent cultures 24 hours, positive colony is obtained, is overexpression citT
Genetic engineering becomes Klebsiella of dwelling.
The present embodiment, which also provides overexpression citT genetic engineerings and becomes the Klebsiella or become of dwelling, dwells Klebsiella
The method that ATCC BAA-830 produce 1,3-PD, comprises the following steps：
Step (1), overexpression citT genetic engineerings is become the Klebsiella or become of dwelling and dwelt Klebsiella ATCC BAA-
The ring of 830 inclined-plane lawn 2 is inoculated in seed culture medium, and Shaking culture obtains seed culture fluid；Wherein, with 1L seed culture mediums
Meter, the seed culture medium includes 10g peptone, 5g dusty yeast, 10g sodium chloride and 20mg chloramphenicol；Shaking culture
Condition be 32 DEG C, rotating speed be that 150rpm, the pH of seed culture medium are 6.8, cultivate 12h.
In step (2), the fermentation tank that seed culture fluid is inoculated in the fermentation medium containing glycerine, culture obtains 1,3-
Propane diols；The volume ratio of the seed culture fluid and the fermentation medium containing glycerine is 0.1：100.
Wherein, in terms of 1L fermentation mediums, the fermentation medium includes 20g glycerine, 10g K2HPO4·3H2O, 2g's
KH2PO4, 1g NH4Cl, 0.1g MgSO4·7H2O, 3g trisodium citrate, 2g yeast extract, 50mg kanamycins and
0.3mL trace element solution, in terms of 1L trace element solutions, the trace element solution includes 7.56g ZnCl2、15.65g
FeCl3·6H2O, 6.28g MnCl2·4H2O, 1.32g CuCl2·2H2O, 6.42g CoCl2·6H2O, 0.48g's
H3BO3With 3.22g Na2MoO4·2H2O；The condition of fermented and cultured is 32 DEG C, rotating speed is that 100rpm, throughput are 0.5vvm, hair
Ferment Medium's PH Value is 6.8, and ferment 32h；
In the 4h of fermentation, 1mM isopropylthiogalactoside (IPTG) induction citT bases are added into fermentation tank
The overexpression of cause, and stream glycerol adding makes glycerol concentration in zymotic fluid control in 40-60g/L during the fermentation.
Using the concentration of 1,3-PD in gas chromatographic detection zymotic fluid, become Klebsiella ATCC BAA-830 hairs of dwelling
The concentration of ferment production 1,3-PD is 52g/L, citT gene of the overexpression from Friedlander's bacillus ATCC 25955
Genetic engineering become the concentration of Klebsiella production 1,3-PD of dwelling into 67g/L, yield is that change is dwelt Klebsiella ATCC
The 129% of BAA-830 yield.
In summary, the engineering strain built using the inventive method, by strengthening the expression of citT genes,
The yield of 1,3- propane diols can be improved.Carry plasmid pDK6-citT and strengthen the Friedlander's bacillus of citT gene expressions
(Klebsiella pneumoniae) ATCC 25955 1,3- propanediols are do not carry plasmid pDK6-citT wild
The 125% of bacterial strain；Carry plasmid pDK7-citT and strengthen the acid-producing Klebsiella bacterium (Klebsiella of citT gene expressions
Oxytoca) ATCC BAA-1236 1,3- propanediols are the 131% of the wild strain for not carrying plasmid pDK7-citT；
The change for carrying plasmid pDK7-citT and strengthening citT expression is dwelt Klebsiella (Klebsiella variicola) ATCC
BAA-830 1,3- propanediols are the 129% of the wild strain for not carrying plasmid pDK7-citT.
Sequence table
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Claims (10)
1. a kind of Klebsiella of overexpression citT genes.
2. the Klebsiella of overexpression citT genes according to claim 1, it is by Klebsiella
Obtained from overexpression citT genes；
Preferably, overexpression during it is by dwelling Klebsiella in Friedlander's bacillus, acid-producing Klebsiella bacterium or change
Obtained from citT genes.
3. the Klebsiella of overexpression citT genes according to claim 1, wherein, the citT genes be from
The citT genes of Klebsiella；
Preferably, the citT genes are from Friedlander's bacillus, acid-producing Klebsiella bacterium or become Klebsiella of dwelling
CitT genes.
4. the Klebsiella of the overexpression citT genes according to Claims 2 or 3, wherein, the kerekou pneumonia primary
Salmonella is Friedlander's bacillus ATCC 25955, and the acid-producing Klebsiella bacterium is acid-producing Klebsiella bacterium ATCC BAA-
1236, change Klebsiella of dwelling is dwelt Klebsiella ATCC BAA-830 into change.
5. a kind of method for the Klebsiella for building the overexpression citT genes described in any one of Claims 1 to 4, the party
Method includes：
CitT genes are connected to expression vector, structure obtains citT overexpression plasmids；
CitT overexpression plasmids are transformed into Klebsiella, cultivated on the resistance culture base of kanamycins or chloramphenicol
Positive colony is obtained, the Klebsiella for the expression citT genes that strengthened.
6. method according to claim 5, wherein, the expression vector includes pDK6 or pDK7.
7. the Klebsiella of the overexpression citT genes described in any one of Claims 1 to 4 is in production 1,3- propane diols
Application.
8. a kind of method for producing 1,3-PD, this method includes：
The Klebsiella of overexpression citT genes described in any one of Claims 1 to 4 is inoculated in seed culture medium,
Shaking culture obtains seed culture fluid；
Seed culture fluid is inoculated in the fermentation medium containing glycerine and cultivated, production obtains 1,3-PD.
9. method according to claim 8, wherein,
The condition of Shaking culture is 32-37 DEG C, rotating speed is that 150-200rpm, the pH of seed culture medium are 6.8-7.0, cultivates 10-
12h；
The condition of fermented and cultured is 32-37 DEG C, rotating speed is that 100-150rpm, throughput are 0.5-1.0vvm, fermentation medium pH
It is worth for 6.5-6.8, fermentation 30-35h；
Preferably, glycerol adding is flowed in fermentation process makes glycerol concentration in zymotic fluid control in 10-60g/L.
10. method according to claim 8, wherein, when fermenting 3-4h, the different of 0.01-1mM is added into zymotic fluid
Propyl dithiocarbamate galactoside induces the overexpression of citT genes.
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Cited By (3)
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|---|---|---|---|---|
| CN109337852A (en) * | 2018-11-12 | 2019-02-15 | 江南大学 | A kind of application recombinating klebsiella in production 1,3- propylene glycol |
| CN109370969A (en) * | 2018-11-12 | 2019-02-22 | 江南大学 | A kind of recombination klebsiella is preparing the application in 1,3-PD |
| WO2021227622A1 (en) * | 2020-05-13 | 2021-11-18 | 内蒙古工业大学 | Klebsiella pneumoniae and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1088886A1 (en) * | 1999-09-30 | 2001-04-04 | Roche Diagnostics GmbH | Methods for production of recombinant Holo-Cytrate Lyase |
| CN101260379A (en) * | 2008-04-23 | 2008-09-10 | 南京工业大学 | Gene engineering bacterium for producing 1,3-propanediol and its preparation method and application |
| CN102199570A (en) * | 2011-03-22 | 2011-09-28 | 清华大学 | Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium |
| CN103484508A (en) * | 2013-09-22 | 2014-01-01 | 北京科技大学 | Method for improving production of L-phenylalanine through L-phenylalanine genetically engineered bacteria |
-
2017
- 2017-08-10 CN CN201710680961.5A patent/CN107325999B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1088886A1 (en) * | 1999-09-30 | 2001-04-04 | Roche Diagnostics GmbH | Methods for production of recombinant Holo-Cytrate Lyase |
| CN101260379A (en) * | 2008-04-23 | 2008-09-10 | 南京工业大学 | Gene engineering bacterium for producing 1,3-propanediol and its preparation method and application |
| CN102199570A (en) * | 2011-03-22 | 2011-09-28 | 清华大学 | Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium |
| CN103484508A (en) * | 2013-09-22 | 2014-01-01 | 北京科技大学 | Method for improving production of L-phenylalanine through L-phenylalanine genetically engineered bacteria |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "MULTISPECIES: anion permease [Klebsiella]，NCBI Reference Sequence: WP_012968326.1", 《GENBANK》 * |
| 李琛: "产1，3-丙二醇基因工程菌的构建", 《中国优秀博硕士学位论文全文数据库 (硕士) 工程科技Ⅰ辑》 * |
| 王勇: "产1，3-丙二醇克雷伯氏杆菌的基因工程改造", 《中国学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337852A (en) * | 2018-11-12 | 2019-02-15 | 江南大学 | A kind of application recombinating klebsiella in production 1,3- propylene glycol |
| CN109370969A (en) * | 2018-11-12 | 2019-02-22 | 江南大学 | A kind of recombination klebsiella is preparing the application in 1,3-PD |
| WO2021227622A1 (en) * | 2020-05-13 | 2021-11-18 | 内蒙古工业大学 | Klebsiella pneumoniae and use thereof |
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