CN109666620A - A kind of engineered strain producing Tagatose, construction method and application - Google Patents
A kind of engineered strain producing Tagatose, construction method and application Download PDFInfo
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- CN109666620A CN109666620A CN201910126820.8A CN201910126820A CN109666620A CN 109666620 A CN109666620 A CN 109666620A CN 201910126820 A CN201910126820 A CN 201910126820A CN 109666620 A CN109666620 A CN 109666620A
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- tagatose
- bacterial strain
- phosphoric acid
- fructose
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- 238000010276 construction Methods 0.000 title claims abstract description 14
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 title claims abstract 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 73
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 42
- 229930091371 Fructose Natural products 0.000 claims abstract description 33
- 239000005715 Fructose Substances 0.000 claims abstract description 32
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 32
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 31
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 27
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 24
- 239000008103 glucose Substances 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 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 claims abstract description 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 11
- 239000005720 sucrose Substances 0.000 claims abstract description 11
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 11
- 108010021582 Glucokinase Proteins 0.000 claims abstract description 10
- 108010073135 Phosphorylases Proteins 0.000 claims abstract description 10
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 claims abstract description 9
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- 230000002708 enhancing effect Effects 0.000 claims abstract description 9
- 102000030595 Glucokinase Human genes 0.000 claims abstract description 8
- 108090000156 Fructokinases Proteins 0.000 claims abstract description 6
- GSXOAOHZAIYLCY-HSUXUTPPSA-N keto-D-fructose 6-phosphate Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@H](O)COP(O)(O)=O GSXOAOHZAIYLCY-HSUXUTPPSA-N 0.000 claims abstract description 6
- 102000009097 Phosphorylases Human genes 0.000 claims abstract description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 3
- 238000000855 fermentation Methods 0.000 claims description 33
- 230000004151 fermentation Effects 0.000 claims description 33
- 238000003259 recombinant expression Methods 0.000 claims description 23
- 101100545272 Caenorhabditis elegans zif-1 gene Proteins 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 15
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- 241000194032 Enterococcus faecalis Species 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 4
- 241000205042 Archaeoglobus fulgidus Species 0.000 claims description 4
- 241000863389 Dictyoglomus thermophilum Species 0.000 claims description 4
- 241000588902 Zymomonas mobilis Species 0.000 claims description 4
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 241000589158 Agrobacterium Species 0.000 claims description 3
- 108090000769 Isomerases Proteins 0.000 claims description 3
- 241000589516 Pseudomonas Species 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 102000004195 Isomerases Human genes 0.000 claims 1
- 230000003834 intracellular effect Effects 0.000 abstract description 5
- 102000003793 Fructokinases Human genes 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 abstract 1
- 230000004153 glucose metabolism Effects 0.000 abstract 1
- 230000037353 metabolic pathway Effects 0.000 abstract 1
- LKDRXBCSQODPBY-OEXCPVAWSA-N D-tagatose Chemical compound OCC1(O)OC[C@@H](O)[C@H](O)[C@@H]1O LKDRXBCSQODPBY-OEXCPVAWSA-N 0.000 description 61
- 229960002737 fructose Drugs 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 16
- 239000007788 liquid Substances 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000029087 digestion Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- 102100031132 Glucose-6-phosphate isomerase Human genes 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
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- 101100175632 Alkalihalobacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) glcK gene Proteins 0.000 description 2
- 101150018370 FRK gene Proteins 0.000 description 2
- 101100174763 Mus musculus Galk1 gene Proteins 0.000 description 2
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- 150000002584 ketoses Chemical group 0.000 description 2
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- 101900154654 Escherichia coli Fructokinase Proteins 0.000 description 1
- 208000007976 Ketosis Diseases 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
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- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 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 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 238000010189 synthetic method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- 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
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01144—Tagatose-6-phosphate kinase (2.7.1.144)
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Abstract
The invention discloses a kind of construction method for producing Tagatose engineered strain and applications.The construction method passes through regulation glucose metabolism intracellular, reduce the enzymatic activity of fructose 6- phosphokinase in cell, enhancing glucokinase and Glucose-6-phosphate isomerase enzymatic activity are to improve the content of fructose 6- phosphoric acid intracellular, the Tagatose route of synthesis being made of 6- phosphoric acid tagatose 3-epimerase and 6- phosphoric acid Tagatose phosphorylase is constructed, the fructose metabolism pathway that enzyme and fructokinase form is penetrated by fructose；Obtain one plant of Corynebacterium glutamicum recombinant bacterial strain; the bacterial strain can be with metabolizable glucose, fructose or Sucrose synthesis Tagatose; compared with the bioconversion fructose reported at present synthesizes Tagatose method, have many advantages, such as it is easy to operate, convenient for separation, suitable large-scale production Tagatose.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of production Tagatose engineering bacteria, the preparation side of the engineering bacteria
Method and the application being used in fermenting and producing Tagatose.
Background technique
Tagatose (D-Tag) is a kind of naturally occurring rare monosaccharide, is the ketose form of galactolipin, and the difference of fructose is to different
Structure body.Sweet taste characteristic is similar to sucrose, and the heat generated is only the one third of sucrose, so being referred to as sweet taste low in calories
Agent.Tagatose have lower calorific value, zero glycemic index, blood glucose passivation, without saprodontia, prebiotic function and anti-oxidant
The excellent nutritive peculiarity such as activity, Tagatose has four big functions: low energy, hypoglycemic, improves intestinal flora and anti-caries tooth.The U.S.
D-Tag is just classified as safety food (GRAS) additive within Food and Drug Administration (FDA) 2003.The safety of Tagatose
Property and its in food application ratified by Japanese health ministry,
Production Tagatose mainly passes through two methods of chemical synthesis and bioconversion at present, and chemical synthesis needs multistep to protect
And deprotection steps, cause energy consumption height, yield low, high production cost.Biotransformation method is using galactolipin as raw material, through L- Arab
Sugared isomery Enzyme catalyzed synthesis D-Tag, it is higher that this method catalyzes and synthesizes efficiency, however galactolipin is expensive, while the reaction
Conversion ratio only has 50%, causes product separation costs to rise, Tagatose high production cost, causes the biotransformation method extensive
Using.Catalysed in vitro system conversion fructose synthesis Tagatose is established in related patents proposition, by phosphorylation and dephosphorylation steps
Conversion ratio is improved, but needs expensive ATP as coenzyme, will cause the rising of Tagatose production cost, is not suitable for extensive
Production, so a kind of low cost is urgently developed, low pollution, the Tagatose new synthetic method of high yield.The present invention proposes to use base
Because engineering technology and micro-biological process construct microorganism recombinant bacterial strain, then it is raw as fermenting substrate using cheap glucose, fructose
The new approaches for producing Tagatose, compared with above method, this method is enriched with environmental-friendly, substrate source and cheap, conversion ratio
Height does not depend on the advantages that coacetylase TP, is suitble to large-scale production Tagatose.
Summary of the invention
The purpose of the present invention one is to provide a kind of construction method for producing Tagatose engineered strain.The construction method includes
Following steps:
(1) enhance the enzymatic activity of glucokinase and Glucose-6-phosphate isomerase, it is characterized in that by introducing strong starting
Son perhaps improves the expression intensity of glucokinase and Glucose-6-phosphate isomerase gene using plasmid expression form or adopts
The higher glucokinase of enzymatic activity and Glucose-6-phosphate isomerase for integrating other source of species with chromosomal integration mode
Gene.
(2) enzymatic activity of fructose 6- phosphokinase is reduced, it is characterized in that using gene knockout or introducing weak promoter side
Formula reduces the expression of cell fructose 6- phosphokinase gene.
(3) enhance the enzymatic activity of 6- phosphoric acid tagatose 3-epimerase and 6- phosphoric acid Tagatose phosphorylase, feature
To use chromosomal integration or plasmid expression form, to introducing 6- phosphoric acid tagatose 3-epimerase intracellular and 6- phosphoric acid tower
Lattice Sugar phosphorylation enzyme gene.
(4) enhancing fructose penetrate enzyme and fructokinase enzymatic activity, it is characterized in that using strong promoter, chromosomal integration or
Person's plasmid expression form, enhancing fructose penetrate the expression of enzyme and fructokinase gene.
The enzymatic activity and reduction fructose 6- phosphokinase of the enhancing glucokinase and Glucose-6-phosphate isomerase
The purpose of enzymatic activity is, improves the content of fructose 6- phosphoric acid intracellular；The enhancing 6- phosphoric acid tagatose epimerase and 6- phosphoric acid
The purpose of the enzymatic activity of Tagatose phosphorylase is fructose 6- phposphate intracellular to be Tagatose 6- phosphoric acid；The enhancing fruit
Sugar is that improving engineered strain utilizes the fructose converting ability for fructose 6- phosphoric acid through the purpose of enzyme and fructokinase.
The construction method of the Tagatose engineered strain is suitable for Corynebacterium glutamicum, Escherichia coli, bacillus subtilis
The bacterial strains such as bacterium, lactic acid bacteria, saccharomyces cerevisiae are that host strain carries out genetic modification, and obtained engineered strain is used for fermentation method synthetic tower lattice
Sugar.
The purpose of the present invention two is to provide the construction method of recombinant bacterial strain Tag, comprising the following steps:
(1) amplification from Agrobacterium tumefaciems Agrobacterium tumefaciens 6- phosphoric acid Tagatose 4- difference to
Isomerase gene (SEQ ID NO:1) and the 6- phosphoric acid ailulose phosphate from Escherichia coli Escherichia coli
Enzyme gene (SEQ ID NO:2), and constructed into expression vector pEC-XK99E, obtain recombinant expression carrier pEC-T6PE-
T6PP1；Amplification derives from the 6- phosphoric acid Tagatose 4- epimerase of thermophilic tennis bacterium Dictyoglomus thermophilum
Gene (SEQ ID NO:3) and 6- phosphoric acid Tagatose phosphorylation from ancient green-ball Pseudomonas Archaeoglobus fulgidus
Enzyme gene (SEQ ID NO:4), and constructed into expression vector pEC-XK99E, obtain recombinant expression carrier pEC-T6PE-
T6PP2 constructs recombinant expression carrier pEC-T6PE-T6PP1 and pEC-T6PE-T6PP2 respectively to wild type glutamic acid rod
In bacterium 13032, recombinant bacterial strain Tag1 and Tag2 are obtained.
(2) in Corynebacterium glutamicum 13032, the fructose 6- phosphorus in Corynebacterium glutamicum is reduced by molecular genetic manipulation
Acid kinase (SEQ ID NO:5) expression, obtains recombinant bacterial strain Tag3.
(3) recombinant expression of 6- phosphoric acid Tagatose 4- epimerase and 6- phosphoric acid Tagatose phosphorylase gene will be carried
Carrier pEC-T6PE-T6PP1 is constructed into recombinant bacterial strain Tag3, obtains recombinant bacterial strain Tag4.
(4) amplification derives from the glucokinase gene (SEQ ID NO:6) and glucose 6- phosphoric acid of Corynebacterium glutamicum
Isomerase gene (SEQ ID NO:7), is constructed in expression vector pXMJ19, obtains recombinant plasmid pXMJ19-GlK-
Recombinant plasmid pXMJ19-GlK-PGI and pEC-T6PE-T6PP1 are converted into recombinant bacterial strain Tag3, obtain recombinant bacterial strain by PGI
Tag5。
(5) amplification penetrates enzyme gene (SEQ ID NO:8) from movement pseudomonad Zymomonas mobilis fructose
With the fructokinase gene (SEQ ID NO:9) of enterococcus faecalis Enterococcus faecalis, and constructed to expression carry
Recombinant vector pEC-T6PE-P6PP-Frk-GlF is obtained in body pEC-T6PE-P6PP, by recombinant plasmid pXMJ19-GlK-PGI and
PEC-T6PE-P6PP-Frk-GlF is constructed into recombinant bacterial strain Tag3, obtains recombinant bacterial strain Tag6.
The purpose of the present invention three is to provide described Corynebacterium glutamicum engineered strain Tag1, Tag2, Tag4, Tag5 and Tag6
Application in Tagatose production.It is characterized in that being synthesized using recombinant bacterial strain Tag1, Tag2, Tag4 and Tag5 glucose fermentation
Tagatose；Recombinant bacterial strain Tag6 can produce tower lattice with glucose fermentation, fructose, sucrose or the mixed liquor of the above three such as molasses
Sugar.
Tagatose is prepared with this method, has many advantages, such as that cost of material is low, yield is high, is suitble to large-scale production Tagatose.
Detailed description of the invention
Fig. 1 fermentation method prepares the technology path of Tagatose.
The high-efficient liquid phase chromatogram that Fig. 2 recombinant bacterial strain Tag5 glucose fermentation synthesizes Tagatose analyzes result.
The high-efficient liquid phase chromatogram of Fig. 3 recombinant bacterial strain Tag6 fermentation fructose synthesis Tagatose analyzes result.
Specific embodiment
The present invention is described in further detail with reference to embodiments.
The percent concentration mentioned in of the invention and embodiment is mass/mass (W/W, unit g/ unless otherwise instructed
100g) percent concentration, mass/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/
100mL) percent concentration.
Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in:
" Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
The material or reagent of same names used are as identical unless otherwise instructed in each embodiment.It is described in embodiment
To various biomaterials acquirement approach be only to provide it is a kind of experiment obtain approach to reach specifically disclosed purpose, do not answer
As limitation when implementing of the invention to biological material source.In fact, the source of used biomaterial is widely, to appoint
Why not the biomaterial that contrary to law and moral ethics can obtain can be replaced according to the prompt in embodiment.
The primer is synthesized by Jiangsu Jin Weizhi Bioisystech Co., Ltd in the present invention.
Embodiment is implemented under the premise of the technical scheme of the present invention, gives detailed embodiment and specific
Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.Ability
Field technique personnel should be understood that without departing from the spirit and scope of the invention can be to the details of technical solution of the present invention
It modifies or replaces with form, but these modifications or substitutions each fall within protection scope of the present invention.
The building of embodiment 1 Corynebacterium glutamicum recombinant bacterial strain Tag1 and Tag2
1. constructing recombinant expression carrier pEC-T6PE-T6PP1
According to the 6- phosphoric acid tower lattice for deriving from Agrobacterium tumefaciems Agrobacterium tumefaciens in KEGG database
Sugared 4- epimerase T6PE gene (SEQ ID NO:1) and from Escherichia coli Escherichia coli 6- phosphoric acid Ah
Lip river ketose phosphorylase T6PP gene (SEQ ID NO:2), design primer 1, primer 2, primer 3 and primer 4, and expanded by PCR
Increase and obtain corresponding sequence, with restriction enzyme SacI and SmaI simultaneously to gene T6PE1 and expression vector pEC-XK99E
(Kirchner O and Tauch A.2003,Tools for genetic engineering in the amino acid-
Producing bacterium Corynebacterium glutamicum.J.Biotechnol.104:287-299) it carries out
Digestion, connection obtain recombinant plasmid pEC-T6PE1；Further using Restriction enzyme Sma I and XbaI simultaneously to gene
T6PP1 and expression vector pEC-T6PE1 carries out digestion, connection obtains recombinant expression carrier pEC-T6PE-T6PP1.Primer sequence
It is as follows:
Primer 1:CAGTCGAGCTCATGACCGCCATTTTGGAAAATC
Primer 2: GATCCCCCGGGTCAGTGGCGGCCTTCGCCCGT
Primer 3:GATCCCCCGGGAAAGGAGGACAACCAtgtcaaccccgcgtcagattcttgctgc a
Primer 4:CTAGCTCTAGATTAACCGAGAAGGTCTTTTGCGGT
2. constructing recombinant expression carrier pEC-T6PE-T6PP2
According to the thermophilic tennis bacterium 6- phosphoric acid tower lattice for deriving from Dictyoglomus thermophilum in ncbi database
Sugared 4- epimerase T6PE gene (SEQ ID NO:3) and from ancient green-ball Pseudomonas Archaeoglobus fulgidus's
6- phosphoric acid Tagatose phosphorylase T6PP gene (SEQ ID NO:4), design primer 5, primer 6, primer 7 and primer 8, and pass through
PCR amplification obtains corresponding sequence, with restriction enzyme SacI and SmaI simultaneously to gene T6PE2 and expression vector pEC-
XK99E carries out digestion, and connection obtains recombinant plasmid pEC-T6PE2；Further simultaneously using Restriction enzyme Sma I and XbaI
Digestion is carried out to gene T6PP2 and expression vector pEC-T6PE2, connection obtains recombinant expression carrier pEC-T6PE-T6PP2.Draw
Object sequence is as follows:
Primer 5:CAGTCGAGCTCatgtggcttagtaaagattatttg
Primer 6:GATCCCCCGGGTTATAAGTTTACAGCATAGTGATAG
Primer 7:GATCCCCCGGGAAAGGAGGACAACCATGTTCAAACCAAAGGCCATCG
Primer 8:CTAGCTCTAGATCACCGCAACAATCCCAGAAACT
2. obtaining Corynebacterium glutamicum recombinant bacterial strain Tag1
By recombinant expression carrier pEC-T6PE-T6PP1 electrotransformation into wild type glutamic acid bar bacterium 13032, weighed
Group bacterial strain Tag1 is obtained by recombinant expression carrier pEC-T6PE-T6PP2 electrotransformation into wild type glutamic acid bar bacterium 13032
Recombinant bacterial strain Tag2.
The building of 2 Corynebacterium glutamicum recombinant bacterial strain Tag4 of embodiment
1. constructing integration vector pK18mobsacB-pfk'
According to the fructose 6- for deriving from Corynebacterium glutamicum Corynebacterium glutamicum in KEGG database
The upstream sequence (SEQ ID NO:10) and downstream sequence (SEQ ID NO:11) of phosphokinase gene, design primer 9, primer
10, primer 11 and primer 12, primer 9 and primer 10 obtain corresponding pfk' gene upstream sequence, primer 9 and primer through PCR amplification
10 obtain corresponding pfk " downstream of gene sequence through PCR amplification；It obtains being made of upstream and downstream sequence using fusion DNA vaccine method
Fusion DNA vaccine segment pfk'-pfk ", further using restriction enzyme EcoRI and HindIII fusion segment pfk'-pfk " and
Carrier pK18mobsacB (A,Tauch A,Jager W,Kal inowski J,Thierbach G,Puhler
A.1994.SmTag mobil izable multi-purpose cloning vectors derived from the
Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the
Chromosome of Corynebacterium glutamicum.Gene 145:69-73) digestion is carried out, connection is obtained
Recombinant plasmid pK18mobsacB-pfk, specific primer sequence are as follows:
Primer 9:ACCGGAATTCATGATTTTGGTTTCCTTCTGCGA
Primer 10:TTCGAATGGAACTTCCTTCAAGCTGGCTGTGCGGACGATTCCT
Primer 11:AGGAATCGTCCGCACAGCCAGCTTGAAGGAAGTTCCATTCGAACG
Primer 12:ACTCAAGCTTGTTAAGACGCAGCTGACCAGTG
2. obtaining the recombinant bacterial strain Tag3 of fructose 6- phosphokinase gene knockout
2.1 preparation 13032 electricity of Corynebacterium glutamicum ATCC turn competent cell, by gene knockout carrier
PK18mobsacB-pfk electrotransformation enters 13032 electricity of Corynebacterium glutamicum ATCC and turns in competent cell.
The positive bacterium colony that 2.2 pickings are grown in that resistant panel of card is lined on the LB plate containing 10% sucrose, is put
It sets in 30 DEG C of incubators and cultivates for 24 hours, the purpose of this step is to be by sucrose lethal, and screening blocks that deletion clone.
2.3 from LB sucrose plate several bacterium colonies of picking, bacterium colony PCR verifying is carried out again with primer 5 and primer 8, through PCR
It is positive colony that amplification, which obtains 1822DNA segment,.
2.4 preservations verify correct bacterial strain, i.e. the recombination glutamic acid rod without fructose 6- phosphokinase gene activity through PCR
Bacterium is named as Tag3.
3. obtaining Corynebacterium glutamicum recombinant bacterial strain Tag4
The recombinant expression for carrying 6- phosphoric acid tagatose 3-epimerase and 6- phosphoric acid Tagatose phosphorylase gene is carried
Body pEC-T6PE-T6PP1 electrotransformation obtains recombinant bacterial strain Tag4 into recombinant bacterial strain Tag3.
The building of 3 Corynebacterium glutamicum recombinant bacterial strain Tag5 of embodiment
1. constructing recombinant expression carrier pXMJ19-GlK-PGI
According in KEGG database derive from Corynebacterium glutamicum glucokinase GlK gene (SEQ ID NO:4) and
Glucose-6-phosphate isomerase pgi gene (SEQ ID NO:5), design primer 13, primer 14, primer 15 and primer 16, and with
Corynebacterium glutamicum gene group is the gene of template PCR amplifications glucokinase gene glk and Glucose-6-phosphate isomerase
Pgi carries out digestion to gene pgi and expression vector pXMJ19 simultaneously with restriction enzyme HindIII and PstI, and is connected with T4
It connects enzyme to be attached, obtains expression vector pXMJ19-PGI；Further using restriction enzyme PstI and XbaI simultaneously to base
Because glk and expression vector pXMJ19-PGI carries out digestion, recombinant expression carrier pXMJ19-GlK-PGI is obtained.Specific primer sequence
It is as follows:
Primer 13:CACTCAAGCTTATGGGATCCATGGCGGACATTTCGACCAC
Primer 14:GACAACTGCAGCTACCTATTTGCGCGGTACCACT
Primer 15:GACAACTGCAGAAAGGAGGACAACCatgccacaaaaaccggccagtt
Primer 16:GATGGTCTAGATTAGTTGGCTTCCACTACAGAGC
2. obtaining Corynebacterium glutamicum recombinant bacterial strain Tag5
By recombinant expression carrier pXMJ19-GlK-PGI and pEC-T6PE-T6PP1 electrotransformation into recombinant bacterial strain Tag3, obtain
Obtain recombinant bacterial strain Tag5.
The building of 4 Corynebacterium glutamicum recombinant bacterial strain Tag6 of embodiment
1. constructing recombinant expression carrier pEC-T6PE-T6PP-Frk-GlF
It is false from movement according to the tuf promoter nucleotide sequence for deriving from Corynebacterium glutamicum in ncbi database
Monad Zymomonas mobilis fructose penetrates enzyme Glf gene order (SEQ ID NO:6), derives from Escherichia coli
Fructokinase Frk gene (SEQ ID NO:7) sequence of Enterococcus faecalis, design primer 17, primer 18 draw
Object 19, primer 20, primer 21 and primer 22, and tuf promoter is obtained using PCR amplification, fructose penetrates enzyme gene glf and fructose
Kinase gene frk segment, using above three segment as template, is obtained tuf-glf-frk and merges segment using fusion DNA vaccine strategy,
And constructed by digestion connection type into recombinant expression plasmid pEC-T6PE-T6PP1, obtain recombinant expression plasmid pEC-
T6PE-T6PP1-FK-GlF。
Primer 17:GATGGTCTAGATGGCCGTTACCCTGCGAATGT
Primer 18:agtacccagatttttccattcaTTGTATGTCCTCCTGGACTTCGTG
Primer 19:CACGAAGTCCAGGAGGACATACAAtgaatggaaaaatctgggtact
Primer 2 0:ACCCTGACTACTTTCAGAACTCATtcacagcgagcgctgaagatcgt
Primer 21:
acgatcttcagcgctcgctgtgaAAAGGAGGACAACCATGAGTTCTGAAAGTAGTCAGGGT
Primer 2 2:CTACTTCTGGGAGCGCCACATCTCCT
2. obtaining Corynebacterium glutamicum recombinant bacterial strain Tag6
By recombinant expression carrier pXMJ19-GlK-PGI and pEC-T6PE-T6PP-Frk-GlF electrotransformation to fructose 6- phosphoric acid
In the recombinant bacterial strain Tag3 that kinase gene knocks out, recombinant bacterial strain Tag6 is obtained.
Application of 5 Corynebacterium glutamicum recombinant bacterial strain Tag1, Tag2, Tag4 and the Tag5 of embodiment in Tagatose production
1, Corynebacterium glutamicum recombinant bacterial strain Tag1 glucose fermentation synthesizes Tagatose
100mL BHI culture medium (the brain heart soaks powder 37g/L, kanamycins 25ng/mL) is selected, final concentration of 2% (matter is added
Amount/volume (W/V, unit g/100mL) percent concentration) glucose, to Corynebacterium glutamicum under the conditions of 30 DEG C, 200rmp
Recombinant bacterial strain Tag1 carries out culture 24-48h, after fermentation, carries out 14000rmp to sample and is centrifuged 20min, and with 0.22 μm
Filtering with microporous membrane, filtrate does high-efficient liquid phase analysis.Efficient liquid phase chromatographic analysis is carried out as follows: instrument is Agilent
High performance liquid chromatograph 1200, analytical column: Sugar-Pak, mobile phase: ultrapure water, flow velocity: 0.4mL/min, column temperature: 80 DEG C, inspection
Device: differential refraction detector is surveyed, applied sample amount is 10 μ l.
2, Corynebacterium glutamicum recombinant bacterial strain Tag2 glucose fermentation synthesizes Tagatose
The cultural method of recombinant bacterial strain Tag2 is identical with 1, add final concentration of 2% in culture medium (mass/volume (W/V,
Unit g/100mL) percent concentration) glucose, cultivate 24-48h, fermentation final sample carry out liquid chromatographic detection.
3, the cultural method of Corynebacterium glutamicum recombinant bacterial strain Tag4 glucose fermentation synthesis Tagatose recombinant bacterial strain Tag4
It is identical with 1, the grape of final concentration of 2% (mass/volume (W/V, unit g/100mL) percent concentration) is added in culture medium
Sugar, cultivates 24-48h, and fermentation final sample carries out liquid chromatographic detection.
3, Corynebacterium glutamicum recombinant bacterial strain Tag5 glucose fermentation synthesizes Tagatose
The cultural method of recombinant bacterial strain Tag5 is identical with 1, add final concentration of 2% in culture medium (mass/volume (W/V,
Unit g/100mL) percent concentration) glucose, cultivate 24-48h, fermentation final sample carry out liquid chromatographic detection.
Fermentation results show that, by 48 hours, recombinant bacterial strain Tag1 can synthesize 1.2g/L Tagatose, weight with glucose fermentation
Group bacterial strain Tag2 can synthesize 0.8g/L Tagatose with glucose fermentation, and recombinant bacterial strain Tag4 can synthesize 4.6g/L with glucose fermentation
Tagatose, compared with recombinant bacterial strain Tag1, tagatose yield improves nearly 3.8 times；Recombinant bacterial strain Tag5 can be closed with glucose fermentation
At 6.4g/L Tagatose, compared with recombinant bacterial strain Tag1, tagatose yield improves 5.3 times.As a result as ((a) indicates fermentation liquid to Fig. 2
0h；(b) fermentation liquid 48h is indicated；(c) Tagatose sterling is indicated).
Application of the 6 Corynebacterium glutamicum recombinant bacterial strain Tag6 of embodiment in Tagatose production
1, Corynebacterium glutamicum recombinant bacterial strain Tag6 glucose fermentation synthesizes Tagatose
The cultural method of recombinant bacterial strain Tag6 is identical with the cultural method of recombinant bacterial strain Tag1 in embodiment 5, in culture medium
The glucose of final concentration of 2% (mass/volume (W/V, unit g/100mL) percent concentration) is added, 24-48h, fermentation are cultivated
Final sample carries out liquid chromatographic detection.
2, Corynebacterium glutamicum recombinant bacterial strain Tag6 fermentation fructose synthesizes Tagatose
The cultural method of recombinant bacterial strain Tag6 is identical with the cultural method of recombinant bacterial strain Tag1 in embodiment 5, in culture medium
The fructose of final concentration of 2% (mass/volume (W/V, unit g/100mL) percent concentration) is added, cultivates 24-48h, fermentation is most
Whole sample carries out liquid chromatographic detection.
3, Corynebacterium glutamicum recombinant bacterial strain Tag6 fermentation Sucrose synthesis Tagatose
The cultural method of recombinant bacterial strain Tag6 is identical with the cultural method of recombinant bacterial strain Tag1 in embodiment 5, in culture medium
Add the sucrose of final concentration of 2% (mass/volume (W/V, unit g/100mL) percent concentration), culture culture 24-48h, hair
Ferment final sample carries out liquid chromatographic detection.
Fermentation results show that, by 48 hours, recombinant bacterial strain Tag6 can synthesize 6.8g/L Tagatose, hair with glucose fermentation
Ferment fructose generates 8.6g/L Tagatose, and fermentation sucrose generates 7.9g/L Tagatose.As a result as ((a) indicates fermentation liquid 0h to Fig. 3；(b)
Indicate fermentation liquid 48h；(c) Tagatose sterling is indicated).
Sequence table
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>a kind of engineered strain for producing Tagatose, its construction method and application
<130> 2018.12.10
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1278
<212> DNA
<213> Agrobacterium tumefaciens
<400> 1
atgaccgcca ttttggaaaa tctcgccgcc gcgcgccgcg ccggcaaacc tgcgggcatc 60
acttcggtct gctcggccca ccccgttgtc ctgcgcgccg caatccgccg cgccgccgcc 120
agtcaaacgg ccgtactgat cgaggccacc tgcaatcagg tcaatcatct cggtggttat 180
accggcatga caccgcgtga cttcgttgcc ttcgtcaaca gcatcgccgc ggaagaagga 240
ctgcccgccg aactgctgat ctttggcggc gatcatctcg gccccaatcc ctggcgcagg 300
gagaaggccg aggacgcgct gacaaaagcc gccgccatgg tcgacgccta tgtcacagct 360
ggttttcgca agatccacct tgatgcatcg atgggctgcg ccggtgagcc ggcagccctg 420
gatgacgtca ccatcgccca ccgcgccgcg aaactcacag ccgttgccga aaaggcagcc 480
actgaggctg gcctgccaaa accgctttat attctgggca ccgaagtgcc ggtgcccggc 540
ggtgccgacc atgtgcttga gaccgtcgca ccgaccgaac cgcaggcggc gcgcaacacc 600
atcgatcttc atcgcgaaat ctttgcgcag cacggtcttt ccgatgcgtt cgaacgggtc 660
atcgcctttg tcgtgcagcc gggtgtggaa ttcggcagcg acaatgtcgt cgcttatgat 720
ccgcaggcag cgcagagcct gagcgccgtg ctggatggcg aaccgcgact ggtcttcgaa 780
gcccattcga ccgattacca gaccgagcct gcccttgcgg cactggtacg cgacggatat 840
ccgatcctca aagttggacc gggcctcacc ttcgcttacc gggaagcgct ttatgcactc 900
gacatgatcg cctccgaaat ggtcggcacc tatggcgacc gaccgctggc gcggactatg 960
gaaaaattga tgttaagcgc gccgggcgac tggcagggcc attaccatgg cgacgacatc 1020
acgctccgat tgcaacgcca ttacagctac agcgaccgca tccgttacta ctggacgcga 1080
ccggaagcgc tcgcggccgt ttccaccttg cataaggcac tggatgggaa gacaattccc 1140
gaaaccctgc tgcgccaata tctcggcgaa ttgccgctcg cggcggttgc gggaaaggaa 1200
ccggaggagg ttctggtcgc ggcggtggat caggtgctgg cgacctatca cgcggcgacg 1260
ggcgaaggcc gccactga 1278
<210> 2
<211> 669
<212> DNA
<213> Escherichia coli
<400> 2
atgtcaaccc cgcgtcagat tcttgctgca atttttgata tggatggatt acttatcgac 60
tcagaacctt tatgggatcg agccgaactg gatgtgatgg caagcctggg ggtggatatc 120
tcccgtcgta acgagctgcc ggacacctta ggtttacgca tcgatatggt ggtcgatctt 180
tggtacgccc ggcaaccgtg gaatgggcca agccgtcagg aagtagtaga acgggttatt 240
gcccgtgcca tttcactggt tgaagagaca cgtccattat taccaggcgt gcgcgaagcc 300
gttgcgttat gcaaagaaca aggtttattg gtgggactgg cctccgcgtc accactacat 360
atgctggaaa aagtgttgac catgtttgac ttacgcgaca gtttcgatgc cctcgcctcg 420
gccgaaaaac tgccttacag caagccgcat ccgcaagtat atctcgactg cgcagcaaaa 480
ctgggcgttg accctctgac ctgcgtagcg ctggaagatt cggtaaatgg catgatcgcc 540
tctaaagcag cccgcatgcg ttccatcgtc gttcctgcgc cagaagcgca aaatgatcca 600
cgttttgtat tagcagacgt caaactttca tcgctgacag aactcaccgc aaaagacctt 660
ctcggttaa 669
<210> 3
<211> 1227
<212> DNA
<213> Dictyoglomus thermophilum
<400> 3
atgtggctta gtaaagatta tttgagaaaa aagggagttt attctatatg tagctctaat 60
ccatatgtga ttgaggcaag tgttgaattt gctaaggaga agaatgatta tattttaatt 120
gaggcgacac ctcatcagat aaaccagttt ggtggatatt caggtatgac tcccgaagat 180
tttaaaaact ttgtaatggg aataataaaa gaaaagggaa tagaagagga tagggtgatt 240
cttggagggg accatttagg ccctctccct tggcaagatg aaccttcttc ttctgcaatg 300
aaaaaggcaa aagaccttat aagggccttt gtggagagtg gttataagaa gatacacctt 360
gattgtagta tgtctctttc tgatgatcct gtagtgctct ctcccgagaa gatagcagaa 420
agggagaggg aacttcttga ggttgcagaa gagactgcta gaaagtacaa ttttcagcct 480
gtgtatgtgg tgggaactga tgtaccggta gctggaggag gcgaagagga aggtattacc 540
tcagtggagg attttagagt agcaatctcc tctttaaaaa aatattttga ggatgttcca 600
aggatatggg ataggataat tggttttgta ataatgcttg gtataggttt taattatgaa 660
aaagtgtttg agtatgacag gattaaggtg agaaaaattt tagaggaggt aaagaaagag 720
aatctttttg ttgaaggtca ctctactgac tatcagacaa aacgtgcatt gagagatatg 780
gtagaggatg gagtaagaat tcttaaggtt ggtcctgctt taacagcaag ttttagaagg 840
ggagtatttt tattaagtag cattgaggat gagcttatat cggaagataa aaggtctaat 900
attaagaaag ttgtgcttga gactatgtta aaagatgata aatattggag aaagtattat 960
aaggattcag aaagattaga attagatatt tggtacaact tacttgatag gattagatat 1020
tattgggaat ataaagagat aaaaatagct ttaaataggc tttttgaaaa tttttcggaa 1080
ggggttgata ttagatacat ctatcaatat ttttatgatt cgtattttaa agtaagagaa 1140
ggaaaaataa gaaatgatcc aagggagcta ataaagaatg aaataaagaa ggtcttggag 1200
gactatcact atgctgtaaa cttataa 1227
<210> 4
<211> 672
<212> DNA
<213> Archaeoglobus fulgidus
<400> 4
atgttcaaac caaaggccat cgcagttgac atagatggca ccctcaccga cagaaagagg 60
gctctgaact gcagggctgt tgaagctctc cgcaaggtaa aaattcccgt gattttggcc 120
actggtaaca tatcttgttt tgcgagggct gcagcaaagc tgattggagt ctcagacgtg 180
gtaatctgcg agaatggggg cgtggtgagg ttcgagtacg atggggagga tattgtttta 240
ggagataaag agaaatgcgt tgaggctgtg agggtgcttg agaaacacta tgaggttgag 300
ctgctggact tcgaatacag gaagtcggaa gtgtgcatga ggaggagctt tgacatcaac 360
gaggcgagaa agctcattga ggggatgggg gttaagcttg tggattcagg ctttgcctac 420
cacattatgg atgctgatgt tagcaaggga aaagctttga agttcgttgc cgagaggctt 480
ggtatcagtt cagcggagtt tgcagttatc ggcgactcag agaacgacat agacatgttc 540
agagttgctg gattcggaat tgctgttgcc aatgccgatg agaggctgaa ggagtatgct 600
gatttagtta cgccatcacc agacggcgag ggggttgttg aggctttgca gtttctggga 660
ttgttgcggt ga 672
<210> 5
<211> 1041
<212> DNA
<213> Corynebacterium glutamicum
<400> 5
atggaagaca tgcgaattgc tactctcacg tcaggcggcg actgccccgg actaaacgcc 60
gtcatccgag gaatcgtccg cacagccagc aatgaatttg gctccaccgt cgttggttat 120
caagacggtt gggaaggact gttaggcgat cgtcgcgtac agctgtatga cgatgaagat 180
attgaccgaa tcctccttcg aggcggcacc attttgggca ctggtcgcct ccatccggac 240
aagtttaagg ccggaattga tcagattaag gccaacttag aagacgccgg catcgatgcc 300
cttatcccaa tcggtggcga aggaaccctg aagggtgcca agtggctgtc tgataacggt 360
atccctgttg tcggtgtccc aaagaccatt gacaatgacg tgaatggcac tgacttcacc 420
ttcggtttcg atactgctgt ggcagtggct accgacgctg ttgaccgcct gcacaccacc 480
gctgaatctc acaaccgtgt gatgatcgtg gaggtcatgg gccgccacgt gggttggatt 540
gctctgcacg caggtatggc cggcggtgct cactacaccg ttattccaga agtacctttc 600
gatattgcag agatctgcaa ggcgatggaa cgtcgcttcc agatgggcga gaagtacggc 660
attatcgtcg ttgcggaagg tgcgttgcca cgcgaaggca ccatggagct tcgtgaaggc 720
cacattgacc agttcggtca caagaccttc acgggaattg gacagcagat cgctgatgag 780
atccacgtgc gcctcggcca cgatgttcgt acgaccgttc ttggccacat tcaacgtggt 840
ggaaccccaa ctgctttcga ccgtgttctg gccactcgtt atggtgttcg tgcagctcgt 900
gcgtgccatg agggaagctt tgacaaggtt gttgctttga agggtgagag cattgagatg 960
atcacctttg aagaagcagt cggaaccttg aaggaagttc cattcgaacg ctgggttact 1020
gcccaggcaa tgtttggata g 1041
<210> 6
<211> 972
<212> DNA
<213> Corynebacterium glutamicum
<400> 6
atgccacaaa aaccggccag tttcgcggtg ggctttgaca tcggcggcac caacatgcga 60
gccgggcttg tcgacgaatc cgggcgcatc gtgaccagtt tgtcggcgcc gtcgccgcgc 120
acgacgcagg caatggaaca ggggattttt gatctagtcg aacagctcaa ggccgaatac 180
ccggttggtg ctgtgggact tgccgtcgcg ggatttttgg atcctgagtg cgaggttgtt 240
cgatttgccc cgcaccttcc ttggcgcgat gagccagtgc gtgaaaagtt ggaaaacctt 300
ttgggcctgc ctgttcgttt ggaacatgat gccaactcag cagcgtgggg tgagcatcgt 360
tttggtgcag ctcaaggcgc tgacaactgg gttttgttgg cactcggcac tggaattggt 420
gcagcgctga ttgaaaaagg cgaaatttac cgtggtgcat atggcacggc accagaattt 480
ggtcatttgc gtgttgttcg tggcggacgc gcatgtgcgt gtggcaaaga aggctgcctg 540
gagcgttact gttccggtac tgccttggtt tacactgcgc gtgaattggc ttcgcatggc 600
tcattccgca acagcgggct gtttgacaag atcaaagccg atccgaattc catcaatgga 660
aaaacgatca ctgcggcagc gcgccaagaa gacccacttg ctctcgccgt tctggaagat 720
ttcagcgagt ggctgggcga aactttggcg atcattgctg atgtccttga cccaggcatg 780
atcatcattg gtggcggact gtccaatgct gccgaccttt atttggatcg ctcggtcaac 840
cactattcca cccgcatcgt cggcgcagga tatcgccctt tggcacgcgt tgccacagct 900
cagttgggtg cggatgctgg catgatcggt gtcgctgatc tagctcgacg ctctgtagtg 960
gaagccaact ag 972
<210> 7
<211> 1623
<212> DNA
<213> Corynebacterium glutamicum
<400> 7
atggcggaca tttcgaccac ccaggtttgg caagacctga ccgatcatta ctcaaacttc 60
caggcaacca ctctgcgtga acttttcaag gaagaaaacc gcgccgagaa gtacaccttc 120
tccgcggctg gcctccacgt cgacctgtcg aagaatctgc ttgacgacgc caccctcacc 180
aagctccttg cactgaccga agaatctggc cttcgcgaac gcattgacgc gatgtttgcc 240
ggtgaacacc tcaacaacac cgaagaccgc gctgtcctcc acaccgcgct gcgccttcct 300
gccgaagctg atctgtcagt agatggccaa gatgttgctg ctgatgtcca cgaagttttg 360
ggacgcatgc gtgacttcgc tactgcgctg cgctcaggca actggttggg acacaccggc 420
cacacgatca agaagatcgt caacattggt atcggtggct ctgacctcgg accagccatg 480
gctacgaagg ctctgcgtgc atacgcgacc gctggtatct cagcagaatt cgtctccaac 540
gtcgacccag cagacctcgt ttctgtgttg gaagacctcg atgcagaatc cacattgttc 600
gtgatcgctt cgaaaacttt caccacccag gagacgctgt ccaacgctcg tgcagctcgt 660
gcttggctgg tagagaagct cggtgaagag gctgtcgcga agcacttcgt cgcagtgtcc 720
accaatgctg aaaaggtcgc agagttcggt atcgacacgg acaacatgtt cggcttctgg 780
gactgggtcg gaggtcgtta ctccgtggac tccgcagttg gtctttccct catggcagtg 840
atcggccctc gcgacttcat gcgtttcctc ggtggattcc acgcgatgga tgaacacttc 900
cgcaccacca agttcgaaga gaacgttcca atcttgatgg ctctgctcgg tgtctggtac 960
tccgatttct atggtgcaga aacccacgct gtcctacctt attccgagga tctcagccgt 1020
tttgctgctt acctccagca gctgaccatg gaatcaaatg gcaagtcagt ccaccgcgac 1080
ggctcccctg tttccactgg cactggcgaa atttactggg gtgagcctgg cacaaatggc 1140
cagcacgctt tcttccagct gatccaccag ggcactcgcc ttgttccagc tgatttcatt 1200
ggtttcgctc gtccaaagca ggatcttcct gccggtgagc gcaccatgca tgaccttttg 1260
atgagcaact tcttcgcaca gaccaaggtt ttggctttcg gtaagaacgc tgaagagatc 1320
gctgcggaag gtgtcgcacc tgagctggtc aaccacaagg tcatgccagg taatcgccca 1380
accaccacca ttttggcgga ggaacttacc ccttctattc tcggtgcgtt gatcgctttg 1440
tacgaacaca tcgtgatggt tcagggcgtg atttgggaca tcaactcctt cgaccaatgg 1500
ggtgttgaac tgggcaaaca gcaggcaaat gacctcgctc cggctgtctc tggtgaagag 1560
gatgttgact cgggagattc ttccactgat tcactgatta agtggtaccg cgcaaatagg 1620
tag 1623
<210> 8
<211> 1427
<212> DNA
<213> Zymomonas mobilis
<400> 8
atggcggaca tttcgaccac ccaggtttgg caagacctga ccgatcatta ctcgctgcta 60
taggcggctt gcttttcggt tacgattcag cggttatcgc tgcaatcggt acaccggttg 120
atatccattt tattgcccct cgtcacctgt ctgctacggc tgcggcttcc ctttctggga 180
tggtcgttgt tgctgttttg gtcggttgtg ttaccggttc tttgctgtct ggctggattg 240
gtattcgctt cggtcgtcgc ggcggattgt tgatgagttc catttgtttc gtcgccgccg 300
gttttggtgc tgcgttaacc gaaaaattat ttggaaccgg tggttcggct ttacaaattt 360
tttgcttttt ccggtttctt gccggtttag gtatcggtgt cgtttcaacc ttgaccccaa 420
cctatattgc tgaaattcgt ccgccagaca aacgtggtca gatggtttct ggtcagcaga 480
tggccattgt gacgggtgct ttaaccggtt atatctttac ctggttactg gctcatttcg 540
gttctatcga ttgggttaat gccagtggtt ggtgctggtc tccggcttca gaaggcctga 600
tcggtattgc cttcttattg ctgctgttaa ccgcaccgga tacgccgcat tggttggtga 660
tgaagggacg tcattccgag gctagcaaaa tccttgctcg tctggaaccg caagccgatc 720
ctaatctgac gattcaaaag attaaagctg gctttgataa agccatggac aaaagcagcg 780
caggtttgtt tgcttttggt atcaccgttg tttttgccgg tgtatccgtt gctgccttcc 840
agcagttagt cggtattaac gccgtgctgt attatgcacc gcagatgttc cagaatttag 900
gttttggagc tgatacggca ttattgcaga ccatctctat cggtgttgtg aacttcatct 960
tcaccatgat tgcttcccgt gttgttgacc gcttcggccg taaacctctg cttatttggg 1020
gtgctctcgg tatggctgca atgatggctg ttttaggctg ctgtttctgg ttcaaagtcg 1080
gtggtgtttt gcctttggct tctgtgcttc tttatattgc agtctttggt atgtcatggg 1140
gccctgtctg ctgggttgtt ctgtcagaaa tgttcccgag ttccatcaag ggcgcagcta 1200
tgcctatcgc tgttaccgga caatggttag ctaatatctt ggttaacttc ctgtttaagg 1260
ttgccgatgg ttctccagca ttgaatcaga ctttcaacca cggtttctcc tatctcgttt 1320
tcgcagcatt aagtatctta ggtggcttga ttgttgctcg cttcgtgccg gaaaccaaag 1380
gtcggagcct ggatgaaatc gaggagatgt ggcgctccca gaagtag 1427
<210> 9
<211> 879
<212> DNA
<213> Enterococcus faecalis
<400> 9
atgacagaaa aacttttagg aagtatcgaa gccggtggca caaaatttgt atgtggcgtt 60
gggacagatg atttgaccat cgtagaacgt gtcagttttc ccacaacaac cccagaagaa 120
acaatgaaaa aagtaataga atttttccaa caatatcctt taaaagcgat tgggattggt 180
tcatttggtc cgattgatat tcacgttgat tctcctacgt atggttatat cacttctaca 240
ccaaaattag cttggcgtaa ctttgacttg ttaggaacta tgaaacaaca ttttgatgtg 300
ccaatggctt ggacaacgga tgtgaatgct gcggcatatg gtgagtatgt tgctggaaat 360
gggcaacata catctagttg tgtatattat acaattggaa ctggtgttgg cgctggagcg 420
attcaaaacg gtgagtttat tgaaggcttt agccacccag aaatggggca tgcgttagtt 480
cgtcgtcatc ctgaagatac gtatgcagga aattgtcctt atcatggaga ttgtttagaa 540
gggattgcag caggaccagc agttgaaggt cgttctggta aaaaaggaca tttattggaa 600
gaggatcata aaacttggga attagaagct tattatttag cgcaagcggc gtacaatacg 660
actttattat tagcgccaga agtgatcatt ttaggtggcg gcgtcatgaa acaacgtcat 720
ttgatgccga aagttcgtga aaaatttgct gaattagtca atggatatgt ggaaacaccg 780
cctttagaaa aatacttggt gacgcctctt ttagaagata atccaggaac aatcggttgc 840
tttgccttgg caaaaaaagc tttaatggct caaaaataa 879
<210> 10
<211> 809
<212> DNA
<213> Corynebacterium glutamicum
<400> 10
aatgattttg gtttccttct gcgagttcgc catgtgactg cggtaaaact gccccggaac 60
cggaatatct cgacgccaca gaacgccatt tgcgggcctt aacaccccgt gggcatacct 120
tttacccatt ccagatattc ggtcgcttaa attgcctagt gtgattccaa acagaaatct 180
ggggcgacct ctaataagag tcgccccgat aagttttttt accgtaatta ttactgggag 240
tcagatactg cgtaagcaat cgcagcagcg ccagcggtca cagtaagaac tgcaggccac 300
gcgccaatct tcttggcaag tgggtgggac aggccaaatg caccaacgta ggttgccagc 360
aggccagtag ctactgcagg acccttcttt tcattccagc ttcgtgcagc aagcgctccg 420
gatgctgcca atggaatggt gcccagtggg cgaatgccgg attcacgggc agtcaaccaa 480
ccgccgatca aacctgctgc gacgacggtg gcagtgctga cctgggatgc ctttttcaat 540
ttcatttcca tggtgagcca gtctagagac aaaatttttc cgcgggggtt ttcttgatct 600
gatccgacaa cccaatgggg gcaaaaatgt gtccgaccaa aaattgtgca gcacaccaca 660
tgcccgctcg gacaatgtcg atttgttaat gaaactgcag ctctggcgat taaataagat 720
ggtcagagac agttttttgg cctgtcaacc cctgtgattc tcttattttt gggtgattgt 780
tccggcgcgg gtgttgtgat gggtttaat 809
<210> 11
<211> 869
<212> DNA
<213> Corynebacterium glutamicum
<400> 11
tttttcgggc ttttatcaac agccaataac agctctttcg cccattgagg tggaggggct 60
gttttttcat gccgtaagga aagtgcaagt aagtgaaatc aagtggccta gatccattga 120
cacttagact gtgacctagg cttgactttc gtgggggagt ggggataagt tcatcttaaa 180
cacaatgcaa tcgattgcat ttacgttcct tatcccacaa taggggtacc ttccagaaag 240
ttggtgagga gatggcttcc gaaacctcca gcccgaagaa gcgggccacc acgctcaaag 300
acatcgcgca agcaacacag ctttcagtca gcacggtgtc ccgggcattg gccaacaacg 360
cgagcattcc ggaatccaca cgcatccgag tggttgaagc cgctcaaaag ctgaactacc 420
gtcccaatgc ccaagctcgt gcattgcgga agtcgaggac agacaccatc ggtgtcatca 480
ttccaaacat tgagaaccca tatttctcct cactagcagc atcgattcaa aaagctgctc 540
gtgaagctgg ggtgtccacc attttgtcca actctgaaga aaacccagag ctgcttggtc 600
agactttggc gatcatggat gaccaacgcc tcgatggaat catcgtggtg ccacacattc 660
agtcagagga acaagtcact gacttggtta acaggggagt gccagtagtg ctggcagacc 720
gtagttttgt taactcgtct attccttcgg ttacctcaga tccagttccg ggcatgactg 780
aagctgtgga cttactcctg gcagctgacg tgcaattggg ctaccttgcc ggcccgcagg 840
atacttccac tggtcagctg cgtcttaac 869
Claims (7)
1. a kind of construction method for producing Tagatose engineered strain, which is characterized in that reduce fructose 6- phosphoric acid in starting strain and swash
The enzymatic activity of enzyme, the enzymatic activity of enhancing 6- phosphoric acid tagatose 3-epimerase, 6- phosphoric acid Tagatose phosphorylase.
2. construction method as described in claim 1, which is characterized in that further include further enhancing glucokinase, glucose
The enzymatic activity of 6- phosphoric acid isomerase.
3. construction method as described in claim 1, which is characterized in that further include further enhancing fructose to swash through enzyme and fructose
The enzymatic activity of enzyme.
4. construction method a method according to any one of claims 1-3, which is characterized in that the starting strain is Corynebacterium glutamicum,
Escherichia coli, bacillus subtilis, lactic acid bacteria, saccharomyces cerevisiae.
5. a kind of construction method of Corynebacterium glutamicum engineered strain Tag, comprising the following steps:
(1) amplification derives from the 6- phosphoric acid Tagatose 4- epimerism of Agrobacterium tumefaciems Agrobacterium tumefaciens
Enzyme gene and 6- phosphoric acid ailulose phosphate enzyme gene from Escherichia coli Escherichia coli, and constructed
Into expression vector pEC-XK99E, recombinant expression carrier pEC-T6PE-T6PP1 is obtained, by recombinant expression carrier pEC-T6PE-
T6PP1 is constructed respectively into wild type glutamic acid bar bacterium 13032, obtains recombinant bacterial strain Tag1；Amplification derives from thermophilic tennis
The 6- phosphoric acid Tagatose 4- epimerism enzyme gene of bacterium Dictyoglomus thermophilum and from ancient green-ball Pseudomonas
The 6- phosphoric acid Tagatose phosphorylase gene of Archaeoglobus fulgidus, and constructed to expression vector pEC-
In XK99E, obtain recombinant expression carrier pEC-T6PE-T6PP2, by recombinant expression carrier pEC-T6PE-T6PP2 construct respectively to
In wild type glutamic acid bar bacterium 13032, recombinant bacterial strain Tag2 is obtained.
(2) in Corynebacterium glutamicum 13032, the fructose 6- phosphoric acid in Corynebacterium glutamicum is reduced by molecular genetic manipulation and is swashed
Enzyme gene expression is horizontal, obtains recombinant bacterial strain Tag3；
(3) recombinant expression carrier of 6- phosphoric acid tagatose 3-epimerase and 6- phosphoric acid Tagatose phosphorylase gene will be carried
PEC-T6PE-T6PP1 is constructed into recombinant bacterial strain Tag3, obtains recombinant bacterial strain Tag4；
(4) amplification derives from the glucokinase and Glucose-6-phosphate isomerase gene of Corynebacterium glutamicum, is constructed
In expression vector pXMJ19, recombinant plasmid pXMJ19-GlK-PGI is obtained, by recombinant plasmid pXMJ19-GlK-PGI and pEC-
T6PE-T6PP1, which is converted into recombinant bacterial strain Tag3, obtains recombinant bacterial strain Tag5；
(5) amplification penetrates enzyme gene and enterococcus faecalis from movement pseudomonad Zymomonas mobilis fructose
The fructokinase gene of Enterococcus faecalis, and constructed into expression vector pEC-T6PE-T6PP1 and obtained
Recombinant vector pEC-T6PE-T6PP1-Frk-GlF, by recombinant vector pXMJ19-GlK-PGI and pEC-T6PE-T6PP1-Frk-
GlF is constructed into recombinant bacterial strain Tag3, obtains recombinant bacterial strain Tag6.
6. recombinant bacterial strain Tag1 described in claim 5, Tag2, Tag4 and Tag5 answering in glucose fermentation synthesis Tagatose
With.
7. application of recombinant bacterial strain Tag6 described in claim 5 in Tagatose synthesis, which is characterized in that recombinant bacterial strain Tag6
Fermentation substrate be one or more of glucose, fructose, sucrose.
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| CN112342178A (en) * | 2020-11-05 | 2021-02-09 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism, preparation method thereof and application thereof in producing tagatose |
| CN112575041A (en) * | 2019-09-30 | 2021-03-30 | 江南大学 | Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium |
| CN112708616A (en) * | 2021-03-29 | 2021-04-27 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilized multienzyme system |
| WO2022148008A1 (en) * | 2021-01-05 | 2022-07-14 | 中国科学院天津工业生物技术研究所 | Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose |
| WO2022213721A1 (en) | 2021-04-07 | 2022-10-13 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilizing multiple enzymes by using artificial oil body |
| WO2022213720A1 (en) | 2021-04-07 | 2022-10-13 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose from biomimetic silicon mineralized microcapsule immobilized multi-enzyme |
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| CN112575041A (en) * | 2019-09-30 | 2021-03-30 | 江南大学 | Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium |
| CN112342178A (en) * | 2020-11-05 | 2021-02-09 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism, preparation method thereof and application thereof in producing tagatose |
| WO2022095684A1 (en) * | 2020-11-05 | 2022-05-12 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism, preparation method therefor, and application of recombinant microorganism in production of tagatose |
| WO2022148008A1 (en) * | 2021-01-05 | 2022-07-14 | 中国科学院天津工业生物技术研究所 | Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose |
| CN112708616A (en) * | 2021-03-29 | 2021-04-27 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilized multienzyme system |
| WO2022206189A1 (en) | 2021-03-29 | 2022-10-06 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilized multi-enzyme system |
| WO2022213721A1 (en) | 2021-04-07 | 2022-10-13 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilizing multiple enzymes by using artificial oil body |
| WO2022213720A1 (en) | 2021-04-07 | 2022-10-13 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose from biomimetic silicon mineralized microcapsule immobilized multi-enzyme |
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