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
- Publication number
- CN108570441A CN108570441A CN201810459120.6A CN201810459120A CN108570441A CN 108570441 A CN108570441 A CN 108570441A CN 201810459120 A CN201810459120 A CN 201810459120A CN 108570441 A CN108570441 A CN 108570441A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- recombined bacillus
- overexpressed
- glutamine
- acetylglucosamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810459120.6A CN108570441A (en) | 2018-05-15 | 2018-05-15 | Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810459120.6A CN108570441A (en) | 2018-05-15 | 2018-05-15 | Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108570441A true CN108570441A (en) | 2018-09-25 |
Family
ID=63572410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810459120.6A Pending CN108570441A (en) | 2018-05-15 | 2018-05-15 | Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108570441A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019191855A1 (en) * | 2018-04-02 | 2019-10-10 | 山东润德生物科技有限公司 | Recombinant bacillus subtilis and use thereof |
| CN110373369A (en) * | 2019-06-25 | 2019-10-25 | 华中农业大学 | A kind of recombined bacillus subtilis engineering bacteria, construction method and its application producing pig glutamine synthelase |
| CN112143751A (en) * | 2020-09-22 | 2020-12-29 | 廊坊梅花生物技术开发有限公司 | Bacillus subtilis engineering bacterium for high nucleoside yield, and construction method and application thereof |
| CN112342230A (en) * | 2019-08-06 | 2021-02-09 | 中国科学院微生物研究所 | Construction and application of engineering strain for producing N-acetylglucosamine |
| WO2022016641A1 (en) * | 2020-07-24 | 2022-01-27 | 上海交通大学 | Strain for producing n-acetylglucosamine, and construction method therefor and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040091976A1 (en) * | 2002-07-01 | 2004-05-13 | Ming-De Deng | Process and materials for production of glucosamine and N-acetylglucosamine |
| CN104928333A (en) * | 2015-07-07 | 2015-09-23 | 江南大学 | Method for knocking out glcK and promoting bacillus subtilis to synthesize acetylglucosamine |
| CN105238724A (en) * | 2015-11-10 | 2016-01-13 | 江南大学 | Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis |
| CN105255803A (en) * | 2015-11-10 | 2016-01-20 | 江南大学 | Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine |
-
2018
- 2018-05-15 CN CN201810459120.6A patent/CN108570441A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040091976A1 (en) * | 2002-07-01 | 2004-05-13 | Ming-De Deng | Process and materials for production of glucosamine and N-acetylglucosamine |
| CN104928333A (en) * | 2015-07-07 | 2015-09-23 | 江南大学 | Method for knocking out glcK and promoting bacillus subtilis to synthesize acetylglucosamine |
| CN105238724A (en) * | 2015-11-10 | 2016-01-13 | 江南大学 | Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis |
| CN105255803A (en) * | 2015-11-10 | 2016-01-20 | 江南大学 | Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine |
Non-Patent Citations (2)
| Title |
|---|
| NC_000913.3: "Escherichia coli str. K-12 substr. MG1655", 《GENBANK》 * |
| 刘延峰: "代谢工程改造枯草芽孢杆菌高效合成 N-乙酰氨基葡萄糖", 《中国博士学位论文全文数据库 工程科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019191855A1 (en) * | 2018-04-02 | 2019-10-10 | 山东润德生物科技有限公司 | Recombinant bacillus subtilis and use thereof |
| CN110373369A (en) * | 2019-06-25 | 2019-10-25 | 华中农业大学 | A kind of recombined bacillus subtilis engineering bacteria, construction method and its application producing pig glutamine synthelase |
| CN110373369B (en) * | 2019-06-25 | 2021-06-08 | 华中农业大学 | Recombinant bacillus subtilis engineering bacterium for producing pig glutamine synthetase, construction method and application thereof |
| CN112342230A (en) * | 2019-08-06 | 2021-02-09 | 中国科学院微生物研究所 | Construction and application of engineering strain for producing N-acetylglucosamine |
| WO2022016641A1 (en) * | 2020-07-24 | 2022-01-27 | 上海交通大学 | Strain for producing n-acetylglucosamine, and construction method therefor and use thereof |
| CN112143751A (en) * | 2020-09-22 | 2020-12-29 | 廊坊梅花生物技术开发有限公司 | Bacillus subtilis engineering bacterium for high nucleoside yield, and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108570441A (en) | Recombined bacillus subtilis and its method for being overexpressed glutamine synthelase promotion synthesis acetylglucosamine | |
| CN104928333B (en) | A kind of method that knockout glcK promotes bacillus subtilis synthesis of acetyl Glucosamine | |
| Yamada et al. | Microbial synthesis of hyaluronan and chitin: New approaches | |
| Cheng et al. | Pullulan: biosynthesis, production, and applications | |
| Göksungur et al. | Production of pullulan from beet molasses and synthetic medium by Aureobasidium pullulans | |
| CN105255803B (en) | A kind of recombined bacillus subtilis for efficiently synthesizing acetylglucosamine | |
| Yuan et al. | Biosynthesis and applications of curdlan | |
| CN106148260B (en) | The recombined bacillus subtilis and its construction method of high yield acetylglucosamine | |
| Yao et al. | Versatile strategies for bioproduction of hyaluronic acid driven by synthetic biology | |
| CN107354119A (en) | A kind of genetic engineering bacterium of high yield hyaluronic acid and its construction method and application | |
| CN107699533A (en) | A kind of recombined bacillus subtilis of acetylglucosamine output increased | |
| CN104498394A (en) | Recombinant bacillus subtilis increased in yield of acetylglucosamine | |
| CN110195036A (en) | A kind of recombination Corynebacterium glutamicum of high yield acetylglucosamine and its application | |
| Yang et al. | Production and optimization of curdlan produced by Pseudomonas sp. QL212 | |
| da Silva et al. | Xanthan: biotechnological production and applications | |
| CN108410783A (en) | A kind of method of high-density cultivation of Escherichia coli fermenting and producing Glucosamine | |
| CN106479945A (en) | A kind of recombined bacillus subtilis efficiently synthesizing acetylglucosamine | |
| Velmurugan et al. | Overexpression of glucose-6-phosphate isomerase in Synechocystis sp. PCC 6803 with disrupted glycogen synthesis pathway improves exopolysaccharides synthesis | |
| CN105176879B (en) | A kind of method that knockout argCJBD improves recombined bacillus subtilis acetylglucosamine yield | |
| CN106367459A (en) | Method for preparing oligomeric hyaluronic acid with different molecular weights | |
| Wu et al. | Synthesis of functional oligosaccharides and their derivatives through cocultivation and cellular NTP regeneration | |
| Wu et al. | A review on polysaccharide biosynthesis in Cordyceps militaris | |
| Zhong et al. | Chemoenzymatic synthesis of fucosylated oligosaccharides using Thermosynechococcus α1–2-fucosyltransferase and their application in the regulation of intestinal microbiota | |
| Wang et al. | Production and characterization of insoluble α-1, 3-linked glucan and soluble α-1, 6-linked dextran from Leuconostoc pseudomesenteroides G29 | |
| WO2007007562A1 (en) | Polysaccharide produced by microorganism belonging to genus bifidobacterium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180925 |