CN108570441A - Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine - Google Patents
Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine Download PDFInfo
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- bacillus subtilis
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 58
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 58
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 title claims abstract description 39
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 27
- 229950006780 n-acetylglucosamine Drugs 0.000 title claims abstract description 27
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 241000588724 Escherichia coli Species 0.000 claims abstract description 16
- 101100031707 Bacillus subtilis (strain 168) nagP gene Proteins 0.000 claims abstract description 7
- 101150070589 nagB gene Proteins 0.000 claims abstract description 7
- 238000003259 recombinant expression Methods 0.000 claims abstract description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 19
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 13
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 235000013619 trace mineral Nutrition 0.000 claims description 12
- 239000011573 trace mineral Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 10
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 claims description 8
- 241001597008 Nomeidae Species 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 229910018890 NaMoO4 Inorganic materials 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 4
- 101150083237 glcK gene Proteins 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 230000002018 overexpression Effects 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 239000012531 culture fluid Substances 0.000 claims description 3
- 230000006801 homologous recombination Effects 0.000 claims description 3
- 238000002744 homologous recombination Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 101150056133 GNPNAT1 gene Proteins 0.000 claims description 2
- 102100023951 Glucosamine 6-phosphate N-acetyltransferase Human genes 0.000 claims description 2
- 101150100121 gna1 gene Proteins 0.000 claims description 2
- 238000010030 laminating Methods 0.000 claims description 2
- 235000000346 sugar Nutrition 0.000 claims description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 abstract description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 abstract description 4
- 229960002442 glucosamine Drugs 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 3
- 239000012530 fluid Substances 0.000 abstract description 3
- 239000006228 supernatant Substances 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 238000012269 metabolic engineering Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 235000001727 glucose Nutrition 0.000 description 9
- MBLBDJOUHNCFQT-LXGUWJNJSA-N aldehydo-N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 6
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 5
- 101000742094 Bacillus subtilis (strain 168) ATP-dependent tyrosine adenylase 2 Proteins 0.000 description 4
- 101001022844 Bacillus subtilis ATP-dependent proline adenylase Proteins 0.000 description 4
- 101000644385 Brevibacillus parabrevis ATP-dependent leucine adenylase Proteins 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 description 2
- 229930189065 blasticidin Natural products 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000009123 feedback regulation Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940025237 fructose 1,6-diphosphate Drugs 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 102000030595 Glucokinase Human genes 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010060820 Joint injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 150000002016 disaccharides Chemical group 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000005067 joint tissue Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
- C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
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Abstract
The invention discloses a kind of recombined bacillus subtilis, on the basis of recombined bacillus subtilis BSGNK, are overexpressed source E. coli glutamine synzyme（EglnA）, obtain recombinant bacterium BSGNKN；The recombined bacillus subtilis BSGNK, be withB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck::lox72For host, respectively with promoter P xylA 、P43Controlglms、GNA1Recombinant expression；It also discloses recombined bacillus subtilis and is overexpressed the method that glutamine synthelase promotes synthesis acetylglucosamine, acetylglucosamine can be improved in the extracellular transformation efficiency accumulated with substrate in obtained recombined bacillus subtilis, acetylglucosamine content is 7.8 g/L in fermented supernatant fluid, it is respectively increased 23.8% compared with compareing bacterium BSGNK, Glucosamine is produced for further metabolic engineering bacillus subtilis to lay a good foundation, and recombined bacillus subtilis construction method is simple, it is easy to use, there is application prospect well.
Description
Technical field
The present invention relates to field of genetic engineering more particularly to a kind of recombination that can improve acetylglucosamine yield are withered
Careless bacillus further relates to be overexpressed glutamine synthelase promotion synthesis acetylamino Portugal using the recombined bacillus subtilis
The method of grape sugar.
Background technology
Acetylglucosamine is a kind of monosaccharide in organism, be widely present in bacterium, yeast, mould, plant and
In animal body.In human body, acetylglucosamine is the synthesis precursor of glycosaminoglycan disaccharide unit, to repairing and maintaining soft
Bone and joint tissue function play an important roll.Therefore, acetylglucosamine is widely used as drug and nutritious food addition
To treat and repair joint injury.In addition, acetylglucosamine also has many applications in cosmetics and pharmaceutical field.Mesh
Before, acetylglucosamine mainly uses chitin in acidolysis shrimp shell or crab shell to produce, and the waste liquid that the method generates is to environment dirt
Contaminate more serious, and obtained product easily causes allergic reaction, and the crowd for being not suitable for seafood allergy takes.
Bacillus subtilis（Bacillus subtilis）It is food safety production bacterial strain, the clear, base with genetic background
It is the important model microorganism of microbial metabolism engineering research because of advantages such as operation maturations.It utilizesB. subtilisSynthesis
When GlcNAc, glucose synthesizes fructose-1, 6-diphosphate under glucokinase and glucose phosphate isomerase effect first, then exists
6- phosphorylated amino glucose synzyme（GlmS）6- phosphorylated amino glucoses are synthesized under catalytic action（GlcN-6P）, then in 6- phosphorus
Sour Glucosamine acetylase（GNA1）Under the action of generate 6- phosphate Glucosamines（GlcNAc-6P）, finally exist
Dephosphorylation generation GlcNAc is transported to extracellular.It includes 3 precursors to integrate building-up process, and respectively fructose-1, 6-diphosphate, acetyl is auxiliary
Enzyme A and glutamine.Studies have shown that the glutamine synthase of bacillus subtilis itself（GlnA）It can be by glutamine
Feedback regulation inhibits glutamine synthase（GlnA）Activity.Therefore, toB. subtilisIn efficiently, excessive synthesis
GlcNAc, it is necessary to release glutamine to glutamine synthase（GlnA）Feedback inhibition.In addition, E. coli glutamine closes
At enzyme（EglnA）It then will not be by the feedback regulation of glutamine.
Invention content
Technical problem to be solved by the invention is to provide one kind passing through strong constitutive promoter P 43 It is overexpressed from big
Enterobacteria glutamine synthelase（EglnA）, glutamine is released to glutamine synthase（GlnA）Feedback inhibition, promote
The recombined bacillus subtilis of extracellular acetylglucosamine accumulation.
In order to solve the above technical problems, the technical scheme is that：Recombined bacillus subtilis, in recombinant bacillus gemma
On the basis of bacillus BSGNK, it is further overexpressed source E. coli glutamine synzyme（EglnA）, obtain recombinant bacterium
BSGNKN；The recombined bacillus subtilis BSGNK, be withB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43Controlglms、GNA1Recombinant expression.
As further preferred technical solution, the E. coli glutamine synzyme（EglnA）Encoding geneglnASuch as geneID in NCBI:Shown in 948370.
As further preferred technical solution, overexpression source E. coli glutamine synzyme（EglnA）
Structure integrant expression frame be SEQID NO.1, and be integrated into the sites glcK through homologous recombination.
The invention further relates to recombined bacillus subtilis to be overexpressed glutamine synthelase promotion synthesis acetamido glucose
The method of sugar crosses table source E. coli glutamine synzyme using recombined bacillus subtilis BSGNK as starting strain
（EglnA）, for producing acetylglucosamine, the recombined bacillus subtilis BSGNK is the recombinant bacterium BSGNKN of gained
WithB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43ControlglmS、GNA1Recombinant expression.
As further preferred technical solution, the recombinant bacterium BSGNKN combinations complex medium laminating production second is utilized
Acylamino- glucose.
The complex medium includes the following components based on g/L as a preferred technical solution,：
Initial glucose 55~65, peptone 5~8, yeast powder 10~14, (NH4)SO45~8, K2HPO4·3H2O 11~
13、KH2PO42~3.5, CaCO33.5~6,8~12ml/L of trace element solution；
The trace element solution includes the following components based on g/L：
MnSO4·5H2O 0.75~1.25, Cocl 2·6H2O 0.3~0.5, NaMoO4·2H2O 0.1~0.3, ZnSO4·
7H2O 0.1~0.3, Alcl3·6H2O 0.05~0.15, Cucl 2H2O 0.05~0.15, H3BO4 0.04~0.06.
As another preferred technical solution, it is produced using the recombinant bacterium BSGNKN combining with fermentation culture medium fermentation method
Acetylglucosamine.
As further preferred technical solution, by the seed culture fluid of the recombined bacillus subtilis after activation with
At least 10% inoculum concentration is transferred in the fermentation medium and is inoculated with, and addition derivant is placed in shaking flask after being inoculated at least 2 h
It is interior, 30~40oTo cultivate at least 48 h under the speed conditions of 200~240rpm in the temperature environment of C.
As further preferred technical solution, the fermentation medium includes the following components based on g/L：
Initial glucose 55~65, peptone 5~8, yeast powder 10~14, (NH4)SO45~8, K2HPO4·3H2O 11~
13、KH2PO42~3.5, CaCO33.5~6,8~12ml/L of trace element solution；
The trace element solution includes the following components based on g/L：
MnSO4·5H2O 0.75~1.25, Cocl 2·6H2O 0.3~0.5, NaMoO4·2H2O 0.1~0.3, ZnSO4·
7H2O 0.1~0.3, Alcl3·6H2O 0.05~0.15, Cucl 2H2O 0.05~0.15, H3BO4 0.04~0.06.
As further preferred technical solution, the derivant includes xylose, and the dosage of the xylose is 5 g/L；Institute
The liquid amount for stating shaking flask is 50 mL.
By adopting the above-described technical solution, the beneficial effects of the invention are as follows：In the base of recombined bacillus subtilis BSGNK
On plinth, it is further overexpressed source E. coli glutamine synzyme（EglnA）, obtained recombined bacillus subtilis can carry
High acetylglucosamine is in the transformation efficiency of extracellular accumulation and substrate, and acetylglucosamine content is in fermented supernatant fluid
7.8 g/L have been respectively increased 23.8% compared with compareing bacterium BSGNK, are produced for further metabolic engineering bacillus subtilis
Glucosamine is laid a good foundation.And recombined bacillus subtilis construction method is simple, is easy to use, and has before applying well
Scape.
Specific implementation mode
With reference to embodiment, the present invention is further explained.In the following detailed description, it is only retouched by way of explanation
Certain exemplary embodiments of the present invention are stated.Undoubtedly, those skilled in the art will recognize, without departing from
In the case of the spirit and scope of the present invention, the described embodiments may be modified in various different ways.Therefore,
Description is regarded as illustrative in nature, and is not intended to limit the scope of the claims.
Embodiment one：
Recombined bacillus subtilis is further overexpressed source Escherichia coli on the basis of recombined bacillus subtilis BSGNK
Glutamine synthelase（EglnA）, obtain recombinant bacterium BSGNKN；The recombined bacillus subtilis BSGNK, be withB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43Controlglms、GNA1Recombinant expression.The E. coli glutamine closes
At enzyme（EglnA）Encoding geneglnASuch as geneID in NCBI:Shown in 948370, geneID in NCBI:948370 be this technology
Content in field known to those of ordinary skill, is no longer described in detail herein.Overexpression source Escherichia coli glutamy
Amine synzyme（EglnA）Structure integrant expression frame be SEQID NO.1, and be integrated into the sites glcK through homologous recombination.
According to announced on NCBI bacillus subtilis (Bacillus subtilis 168It is micro- purchased from U.S. typical case
Biological deposits center, ATCC No.27370) integration site glcK upstream and downstream sequence, the sequence of blasticidin resistance gene
Row, strong constitutive promoter P43, and derive from E. coli glutamine synzyme（EglnA）Build sequence such as SEQ ID
Frame is integrated shown in NO.1.
The integration frame built is converted into recombined bacillus subtilis BSGNK, passes through blasticidin resistance plate screening, bacterium
PCR verifications are fallen, successful integration is confirmed, obtains recombined bacillus subtilis BSGNKN.Recombined bacillus subtilis BSGNK is to pass through
pP43NMK-GNA1The free expression of plasmidGNA1Gene passes through pM7Z6M-P xylA -glmSPlasmid integration is expressedglmSGene.Weight
The construction method of group bacillus subtilis BSGNK is referring to document Liu, Y.et al. Modular pathway
engineering of Bacillus subtilis for improved N-acetylglucosamine production.
Metab. Eng. 23:42-52, 2014。
Recombined bacillus subtilis is overexpressed glutamine synthelase and promotes to synthesize acetylglucosamine in the present embodiment
Method cross table source E. coli glutamine synzyme using recombined bacillus subtilis BSGNK as starting strain
（EglnA）, for producing acetylglucosamine, the recombined bacillus subtilis BSGNK is the recombinant bacterium BSGNKN of gained
WithB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43ControlglmS、GNA1Recombinant expression.
It is specific to utilize recombinant bacterium BSGNKN for producing acetylglucosamine as the composite algorithm in conjunction with complex medium.Institute
It includes the following components based on g/L to state complex medium：
Initial glucose 60, peptone 6, yeast powder 12, (NH4)SO4 6、 K2HPO4·3H2O 12.5、KH2PO4 2.5、
CaCO35,10 ml/L of trace element solution；The trace element solution includes the following components based on g/L：MnSO4·5H2O
1、Cocl 2·6H2O 0.4、NaMoO4·2H2O 0.2、ZnSO4·7H2O 0.2、 Alcl3·6H2O 0.1、Cucl 2·H2O
0.1、H3BO4 0.05。
Compound criteria condition：By 37oC, the seed of 8 h is cultivated under 220 rpm to be transferred to again with 3% inoculum concentration
Culture medium is closed, 5 g/L of derivant xylose is added after being inoculated with 2 h, in 500 mL shaking flasks, 37oC, it is cultivated under the conditions of 220rpm
48 h, shaking flask liquid amount are 50 mL.
Embodiment two：
The present embodiment produces acetylglucosamine using the recombinant bacterium BSGNKN combining with fermentation culture medium fermentation method.Specifically
For the seed culture fluid of the recombined bacillus subtilis after activation is transferred to the fermentation at least 10% inoculum concentration and is trained
It supports and is inoculated in base, addition derivant is placed in 500 mL shaking flasks after being inoculated at least 2 h, 37oIn the temperature environment of C with
At least 48 h are cultivated under the speed conditions of 220rpm.The derivant includes xylose, and the dosage of the xylose is 5 g/L；Institute
The liquid amount for stating shaking flask is 50 mL.
The fermentation medium of the present embodiment includes the following components based on g/L：
Initial glucose 60, peptone 6, yeast powder 12, (NH4)SO4 6、 K2HPO4·3H2O 12.5、KH2PO4 2.5、
CaCO35,10 ml/L of trace element solution；The trace element solution includes the following components based on g/L：MnSO4·5H2O
1、Cocl 2·6H2O 0.4、NaMoO4·2H2O 0.2、ZnSO4·7H2O 0.2、 Alcl3·6H2O 0.1、Cucl 2·H2O
0.1、H3BO4 0.05。
By the above method, acetylglucosamine can be improved at extracellular accumulation and bottom in obtained recombined bacillus subtilis
The transformation efficiency of object, acetylglucosamine content is 7.8 g/L in fermented supernatant fluid, is carried respectively compared with compareing bacterium BSGNK
It is high by 23.8%, realize raising of the acetylglucosamine in the extracellular yield of recombined bacillus subtilis.
N acetylglucosamine n synthesis, thalli growth, by-product synthesis and yield of the present invention are compared with compareing bacterium BSGNK such as table
Shown in 1：
Table 1
Although the present invention is disclosed with preferred embodiment, it is not limited to the present invention, any person skilled in the art,
It does not depart from the spirit and scope of the present invention, can all do various change and modification, therefore protection scope of the present invention should be with
Subject to claims are defined.
Sequence table
<110>The Nature biology Group Co., Ltd
<120>Recombined bacillus subtilis and its side for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine
Method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4252
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis)
<400> 1
catcaggagc catattcgga tgcttaggtg cattactgta tgttgctttg tctaatcgaa 60
aaatgttttt aagaactata ggaaccaata ttattgtcat tattatcatc aatctgggct 120
ttggttttgc ggtttcgaat attgataatt caggacatat cggcggcttg attggcggct 180
ttttcgctgc agcagcactg gggttgccta aagccggagc ctttggaaaa agattgttgt 240
cagcggtttt gctgattgct ttggctgttg gatttttata ttacggattg cattcgcctt 300
cacaccagga gtcagcgctg attcagcagg caagcgagct gtatcaggaa ggaaagtatg 360
aagaagtaac agaattgctg aacggagaag cagcgcaaaa agatgcctct gccgatcttt 420
tgaaaattct tgctgtttct gatattcaaa tcggcgaata tgatcaggcg gtttcccttt 480
tggaacgagc agtgaaaaaa gaacctaaag accatgcttc ctattacaat ttagcgttat 540
tgtatgcgga aaaaaatgag cttgcacaag cagaaaaggc catccagacg gctgtgaaac 600
tgaagccgaa ggagcagcgc tacaaggagc tgcagcggca aattgaaaac aataaagaat 660
catagaggtg gaatatggaa aggaagctgg cttttttgtc agcgttttct tcttgactga 720
ccgcttcctt atgaatacgc ttatgatagt taagttcaag aaatggtgag gaaaatgaat 780
acattttatg atgtgcagca gctgttaaaa acgtttggcc acattgttta ttttggagac 840
agagagcttg aaattgagtt tatgctggat gaattaaagg aattatacat gaaccacatg 900
attgaaaagg agcagtgggc cagagcggca gctgtccttc gaaaagaatt ggaacaaaca 960
aaaaacggaa gagattttta taaaggctaa ggtgataaaa gaattcgagc tcggtacccg 1020
gggatcctct agagataccg ttcgtatagc atacattata cgaagttatc ttgatatggc 1080
tttttatatg tgttactcta catacagaaa ggaggaacta aacatggcca agttgaccag 1140
tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 1200
gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 1260
gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt 1320
gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 1380
ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt 1440
cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaat 1500
aacttcgtat agcatacatt atacgaacgg taaatcgtcg actgataggt ggtatgtttt 1560
cgcttgaact tttaaataca gccattgaac atacggttga tttaataact gacaaacatc 1620
accctcttgc taaagcggcc aaggacgccg ccgccggggc tgtttgcgtt cttgccgtga 1680
tttcgtgtac cattggttta cttatttttt tgccaaggct gtaatggctg aaaattctta 1740
catttatttt acatttttag aaatgggcgt gaaaaaaagc gcgcgattat gtaaaatata 1800
aagtgatagc ggtaccatta taggtaagag aggaatgtac acatgtccgc tgaacacgta 1860
ctgacgatgc tgaacgagca cgaagtgaag tttgttgatt tgcgcttcac cgatactaaa 1920
ggtaaagaac agcacgtcac tatccctgct catcaggtga atgctgaatt cttcgaagaa 1980
ggcaaaatgt ttgacggctc ctcgattggc ggctggaaag gcattaacga gtccgacatg 2040
gtgctgatgc cagacgcatc caccgcagtg attgacccgt tcttcgccga ctccaccctg 2100
attatccgtt gcgacatcct tgaacctggc accctgcaag gctatgaccg tgacccgcgc 2160
tccattgcga agcgcgccga agattacctg cgttccactg gcattgccga caccgtactg 2220
ttcgggccag aacctgaatt cttcctgttc gatgacatcc gtttcggatc atctatctcc 2280
ggttcccacg ttgctatcga cgatatcgaa ggcgcatgga actcctccac ccaatacgaa 2340
ggtggtaaca aaggtcaccg tccggcagtg aaaggcggtt acttcccggt tccaccggta 2400
gactcggctc aggatattcg ttctgaaatg tgtctggtga tggaacagat gggtctggtg 2460
gttgaagccc atcaccacga agtagcgact gctggtcaga acgaagtggc tacccgcttc 2520
aataccatga ccaaaaaagc tgacgaaatt cagatctaca aatatgttgt gcacaacgta 2580
gcgcaccgct tcggtaaaac cgcgaccttt atgccaaaac cgatgttcgg tgataacggc 2640
tccggtatgc actgccacat gtctctgtct aaaaacggcg ttaacctgtt cgcaggcgac 2700
aaatacgcag gtctgtctga gcaggcgctg tactacattg gcggcgtaat caaacacgct 2760
aaagcgatta acgccctggc aaacccgacc accaactctt ataagcgtct ggtcccgggc 2820
tatgaagcac cggtaatgct ggcttactct gcgcgtaacc gttctgcgtc tatccgtatt 2880
ccggtggttt cttctccgaa agcacgtcgt atcgaagtac gtttcccgga tccggcagct 2940
aacccgtacc tgtgctttgc tgccctgctg atggccggtc ttgatggtat caagaacaag 3000
atccatccgg gcgaagccat ggacaaaaac ctgtatgacc tgccgccaga agaagcgaaa 3060
gagatcccac aggttgcagg ctctctggaa gaagcactga acgaactgga tctggaccgc 3120
gagttcctga aagccggtgg cgtgttcact gacgaagcaa ttgatgcgta catcgctctg 3180
cgtcgcgaag aagatgaccg cgtgcgtatg actccgcatc cggtagagtt tgagctgtac 3240
tacagcgtct aaaattgtgt aaatgaaatt gattttttgt tgtgctcagg ttaagattta 3300
atttgatgtg ttaatgagaa tgttgggaat agactgattt ttttgagcgt gctgcatagg 3360
aggttgaaat gcgaaaaacg tttttttcga agatttcatt tatgctgatt gccattttat 3420
tgatgtggct gaaaacgtat gctgtttaca aaaccagttt tcatattaaa atcgacaatc 3480
taacacagga atttattctg tttatcaacc cattgagttt tttgttgctt atttttggcc 3540
tcagcctgtt tttaaaaggc aaaaacagaa atcgctacat tatcgcgatg agctgtcttg 3600
tcacgtttgt attgctggca aatatggttt tttaccgttt ttacaatgat ttcttaacaa 3660
tccctgttct ttttcaaacg agcaatatgg gtgatctcgg aagcagcatc ggaacacttc 3720
ttgagccgac agacctccta ttagctgtag atattgcggt tttaatatgg cttcacatcc 3780
ggcaaaaagc ttttcaatcg gacattccct caacgaaaaa tgaacgggcg gcttattttt 3840
tgttcgttgc ttctgtttat ttcttcaacc tgggcttgtc tgaggcggaa agacctcagc 3900
tattgacacg ctcatttgac agagaaatgc ttgttaaaaa cattagcctg tttaattttc 3960
atatttacga tggcgttctt cagtcaaagc aatccgcaca gagagcgttg gcagacagca 4020
acagcctgac ggagattgaa aactacgtaa ccgctaatgc gaaggatgcc aacaaacgct 4080
tattcggcgc tgcaaaagga aggaacgtca ttctcgtatc cttagagtcg acgcaaagct 4140
tcgtgattaa tgaaaaattg aatggagaag aaatcacgcc ttttctgaat gactttataa 4200
aacagagcta caactttaat aatgtttacc accaaacagg ccaggggaaa ac 4252
Claims (10)
1. recombined bacillus subtilis, it is characterised in that：It is further to cross table on the basis of recombined bacillus subtilis BSGNK
Up to source E. coli glutamine synzyme（EglnA）, obtain recombinant bacterium BSGNKN；The recombined bacillus subtilis
BSGNK, be withB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43Controlglms、GNA1Recombinant expression.
2. recombined bacillus subtilis as described in claim 1, it is characterised in that：The E. coli glutamine synzyme
（EglnA）Encoding geneglnASuch as geneID in NCBI:Shown in 948370.
3. recombined bacillus subtilis as claimed in claim 2, it is characterised in that：Overexpression source Escherichia coli paddy
Glutamine synzyme（EglnA）Structure integrant expression frame be SEQID NO.1, and be integrated into the sites glcK through homologous recombination.
4. being overexpressed glutamine synthelase using recombined bacillus subtilis as claimed in claim 1,2 or 3 promotes synthesis
The method of acetylglucosamine, it is characterised in that：Using recombined bacillus subtilis BSGNK as starting strain, table source is crossed
E. coli glutamine synzyme（EglnA）, the recombinant bacterium BSGNKN of gained is used to produce acetylglucosamine, described heavy
Group bacillus subtilis BSGNK be withB. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72For host, respectively with promoter P xylA 、P43ControlglmS、GNA1's
Recombinant expression.
5. recombined bacillus subtilis as claimed in claim 4, which is overexpressed glutamine synthelase, promotes synthesis acetylamino Portugal
The method of grape sugar, it is characterised in that：Utilize the recombinant bacterium BSGNKN combinations complex medium laminating production acetamido glucose
Sugar.
6. recombined bacillus subtilis as claimed in claim 5, which is overexpressed glutamine synthelase, promotes synthesis acetylamino Portugal
The method of grape sugar, it is characterised in that：The complex medium includes the following components based on g/L：
Initial glucose 55~65, peptone 5~8, yeast powder 10~14, (NH4)SO45~8, K2HPO4·3H2O 11~
13、KH2PO42~3.5, CaCO33.5~6,8~12ml/L of trace element solution；
The trace element solution includes the following components based on g/L：
MnSO4·5H2O 0.75~1.25, Cocl 2·6H2O 0.3~0.5, NaMoO4·2H2O 0.1~0.3, ZnSO4·
7H2O 0.1~0.3, Alcl3·6H2O 0.05~0.15, Cucl 2H2O 0.05~0.15, H3BO4 0.04~0.06.
7. recombined bacillus subtilis as claimed in claim 4, which is overexpressed glutamine synthelase, promotes synthesis acetylamino Portugal
The method of grape sugar, it is characterised in that：Utilize recombinant bacterium BSGNKN combining with fermentation culture medium fermentation method production acetylamino Portugal
Grape sugar.
8. recombined bacillus subtilis as claimed in claim 7, which is overexpressed glutamine synthelase, promotes synthesis acetylamino
The method of glucose, it is characterised in that：By the seed culture fluid of the recombined bacillus subtilis after activation at least 10%
Inoculum concentration is transferred in the fermentation medium and is inoculated with, and is inoculated with after at least 2 h and derivant is added is placed in shaking flask, 30~
40 oTo cultivate at least 48 h under the speed conditions of 200~240rpm in the temperature environment of C.
9. recombined bacillus subtilis as claimed in claim 8, which is overexpressed glutamine synthelase, promotes synthesis acetylamino Portugal
The method of grape sugar, it is characterised in that：The fermentation medium includes the following components based on g/L：
Initial glucose 55~65, peptone 5~8, yeast powder 10~14, (NH4)SO45~8, K2HPO4·3H2O 11~
13、KH2PO42~3.5, CaCO33.5~6,8~12ml/L of trace element solution；
The trace element solution includes the following components based on g/L：
MnSO4·5H2O 0.75~1.25, Cocl 2·6H2O 0.3~0.5, NaMoO4·2H2O 0.1~0.3, ZnSO4·
7H2O 0.1~0.3, Alcl3·6H2O 0.05~0.15, Cucl 2H2O 0.05~0.15, H3BO4 0.04~0.06.
10. recombined bacillus subtilis as claimed in claim 8, which is overexpressed glutamine synthelase, promotes synthesis acetylamino
The method of glucose, it is characterised in that：The derivant includes xylose, and the dosage of the xylose is 5 g/L；The dress of the shaking flask
Liquid measure is 50 mL.
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