CN108546668A - Recombined bacillus subtilis and its overexpression 6- phosphorylated amino glucose synzyme promote acetylglucosamine synthetic method - Google Patents
Recombined bacillus subtilis and its overexpression 6- phosphorylated amino glucose synzyme promote acetylglucosamine synthetic method Download PDFInfo
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Abstract
The invention discloses a kind of recombined bacillus subtilis, on the basis of recombined bacillus subtilis BSGNK, are further overexpressed 6 phosphorylated amino glucose synzyme（GlmS）, obtain recombinant bacterium BSGNKS；The recombined bacillus subtilis BSGNK is with 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δs glck of B. subtilis::Lox72 is host, respectively with promoter PxylA、P43Control the recombinant expression of glms, GNA1；It also discloses recombined bacillus subtilis and is overexpressed 6 phosphorylated amino glucose synzyme promotion acetylglucosamine synthetic method, acetylglucosamine can be improved in the extracellular transformation efficiency accumulated with substrate in obtained recombined bacillus subtilis, acetylglucosamine content is 11.4 g/L in fermented supernatant fluid, it is respectively increased 37.4% 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, convenient for promoting the use of.
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 6- phosphorylated amino glucose synzyme promotion synthesis second using the recombined bacillus subtilis
The method of acylamino- glucose.
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) is that one kind being widely used as Food enzyme and important nutrient laden
The production host of product, product are " generally regarded as safe " (GRAS) safety level by FDA certifications
Not.The recombined bacillus subtilis built in the past（BSGNK）Fermenting and producing acetylglucosamine（GlcNAc）When, exist
The problem of GlcNAc low outputs.In B. subtilis, glucose is made in glucokinase and glucose phosphate isomerase first
With lower synthesis fructose-1, 6-diphosphate, then in 6- phosphorylated amino glucose synzyme（GlmS）6- phosphorylated aminos are synthesized under catalytic action
Glucose（GlcN-6P）, then in 6- phosphorylated amino glucose acetylases（GNA1）Under the action of generate 6- phosphate amino
Glucose（GlcNAc-6P）, finally it is transported in dephosphorylation generation GlcNAc extracellular.But some researches show that fructose-1, 6-diphosphates
6- phosphorylated amino glucoses are generated under the catalytic action of GlmS（GlcN-6P）Process by by glmS ribozymes switch mediate
Strict-feedback controls, and detailed process is as follows：When intracellular GlcN-6P is accumulated, it can switch and combine with glmS ribozymes, activate core
Enzyme switch carries out Self cleavage to glmS gene transcripts；3 ' end mRNA cut portions are because the 5 ' ends-OH of cutting formation are by RNA enzyme
J1 specific recognitions, mRNA is further degraded under RNA enzyme J1 effects, is caused the mRNA of glmS genes that can not translate, is reduced
The expression of glmS genes；And when GlcN6P concentration is relatively low, glmS gene transcripts can be translated normally.Therefore, to excess
Synthesize GlcNAc, it is necessary to release glmS ribozymes and regulate and control in the inhibition of transcriptional level to GlcNAc synthesis key enzyme GlmS.
Invention content
Technical problem to be solved by the invention is to provide one kind passing through strong constitutive promoter P43It is overexpressed 6- phosphoric acid ammonia
Base Glucose Synthetase（GlmS）, it releases glmS ribozymes and GlcNAc synthesis key enzyme GlmS is regulated and controled in the inhibition of transcriptional level,
The mRNA for avoiding glmS genes is degraded by RNA enzyme so that the mRNA of glmS genes is normally translated, and promotes extracellular acetylamino
The recombined bacillus subtilis of grape sugar 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 6- phosphorylated amino glucose synzyme（GlmS）, obtain recombinant bacterium BSGNKS；
The recombined bacillus subtilis BSGNK is with 168 Δ nagP Δ gamP Δ gamA Δs nagA of B. subtilis
Δ nagB Δ ldh Δ pta Δ glck ::Lox72 is host, respectively with promoter PxylA、P43Control glms, GNA1
Recombinant expression.
As further preferred technical solution, the 6- phosphorylated amino glucoses synzyme（GlmS）Encoding gene glmS
Such as geneID in NCBI:Shown in 938736.
As further preferred technical solution, the 6- phosphorylated amino glucoses synzyme（GlmS）Structure integrate table
It is SEQID NO.1 up to frame, and the sites glcK is integrated into through homologous recombination.
The invention further relates to recombined bacillus subtilis to be overexpressed 6- phosphorylated amino glucose synzyme promotion acetyl ammonia
Base glucose synthetic method is overexpressed 6- phosphorylated amino glucoses using recombined bacillus subtilis BSGNK as starting strain
Synzyme（GlmS）, the recombinant bacterium BSGNKS of gained is for producing acetylglucosamine, the recombined bacillus subtilis
BSGNK is with 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δs of B. subtilis
pta Δ glck ::Lox72 is host, respectively with promoter PxylA、P43Control the recombinant expression of glms, GNA1.
As further preferred technical solution, the recombinant bacterium BSGNKS combinations complex medium laminating production second is utilized
Acylamino- glucose.
As further preferred technical solution, 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.
As further preferred technical solution, produced using the recombinant bacterium BSGNKS 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 6- phosphorylated amino glucose synzyme（GlmS）, second can be improved in obtained recombined bacillus subtilis
Acylamino- glucose acetylglucosamine content in the extracellular transformation efficiency accumulated with substrate, fermented supernatant fluid is 11.4
G/L has been respectively increased 37.4% compared with compareing bacterium BSGNK, and amino is produced for further metabolic engineering bacillus subtilis
Glucose is laid a good foundation, and recombined bacillus subtilis construction method is simple, is easy to use, and has application prospect well.
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 6- phosphorylated aminos Portugal on the basis of recombined bacillus subtilis BSGNK
Grape sugar synzyme（GlmS）, obtain recombinant bacterium BSGNKS；The recombined bacillus subtilis BSGNK is with B. subtilis
168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck ::Lox72 is
Host, respectively with promoter PxylA、P43Control the recombinant expression of glms, GNA1.The 6- phosphorylated amino glucoses synzyme
（GlmS）GeneID in encoding gene glmS such as NCBI:Shown in 938736, geneID in the NCBI:938736 lead for this technology
Content in domain known to those of ordinary skill, is no longer described in detail herein.The 6- phosphorylated amino glucoses synzyme
（GlmS）Structure integrant expression frame be SEQID NO.1, and be integrated into the sites glcK through homologous recombination.
According to the bacillus subtilis announced on NCBI, (Bacillus subtilis 168 are 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 P43And 6- phosphorylated amino glucose synzyme（GlmS）Sequence is built as shown in SEQ ID NO.1
Integration frame.
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 BSGNKS.The structure of recombined bacillus subtilis BSGNK
Method 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。BSGNK
It is that pM7Z6M-P is passed through by the free expression GNA1 genes of pP43NMK-GNA1 plasmidsxylA- glmS plasmid integrations express glmS
Gene.
Recombined bacillus subtilis is overexpressed 6- phosphorylated amino glucose synzyme and promotes acetylglucosamine synthesis
Method is overexpressed 6- phosphorylated amino glucose synzyme using recombined bacillus subtilis BSGNK as starting strain（GlmS）,
For the recombinant bacterium BSGNKS of gained for producing acetylglucosamine, the recombined bacillus subtilis BSGNK is with B.
subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck ::
Lox72 is host, respectively with promoter PxylA、P43Control the recombinant expression of glms, GNA1.It is tied using the recombinant bacterium BSGNKS
Close complex medium laminating production acetylglucosamine.
Wherein, 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, micro-
8~12ml/L of secondary 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.
Specifically contain：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；Trace element solution contains：MnSO4·5H2O 1.0、
Cocl 2·6H2O 0.4、NaMoO4·2H2O 0.2、ZnSO4·7H2O 0.2、 Alcl3·6H2O 0.1、Cucl 2·H2O
0.1 and 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.Seed culture medium (g/L)：Tryptone 10, yeast powder 5, NaCl 10.
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 shaking flask after being inoculated at least 2 h, 30~40oIn the temperature environment of C with
At least 48 h are cultivated under the speed conditions of 200~240rpm.Seed culture medium includes point based on g/L with the following group：Tryptose
Peptone 10, yeast powder 5, NaCl 10.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.
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, micro-
8~12ml/L of 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.
Specifically, 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；Trace element solution contains based on g/L：MnSO4·5H2O
1.0、Cocl 2·6H2O 0.4、NaMoO4·2H2O 0.2、ZnSO4·7H2O 0.2、 Alcl3·6H2O 0.1、Cucl 2·
H2O 0.1 and H3BO4 0.05。
The assay method of acetylglucosamine：
High performance liquid chromatography (HPLC) detection method：Agilent1200, RID detector, NH2Column (250 × 4.6mm, 5 μm), stream
Dynamic phase：70% acetonitrile, 0.75 mL/min of flow velocity, column temperature 30oC, sampling volume are 10 μ L.Concentration of glucose in zymotic fluid
Detection：SBA bio-sensing analyzers.
On the basis of recombined bacillus subtilis BSGNK, it is further overexpressed 6- phosphorylated amino glucose synzyme
（GlmS）, obtained recombined bacillus subtilis can be improved acetylglucosamine extracellular accumulation and substrate transformation efficiency,
Acetylglucosamine content is 11.4 g/L in fermented supernatant fluid, has been respectively increased 37.4% compared with compareing bacterium BSGNK, has been
Further metabolic engineering bacillus subtilis production Glucosamine is laid a good foundation, and recombined bacillus subtilis is built
Method is simple, is easy to use, and has application prospect well.
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 overexpression 6- phosphorylated amino glucose synzyme promote acetylglucosamine
Synthetic method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4645
<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 acatgtgtgg aatcgtaggt 1860
tatatcggtc agcttgatgc gaaggaaatt ttattaaaag ggttagagaa gcttgagtat 1920
cgcggttatg actctgctgg tattgctgtt gccaacgaac agggaatcca tgtgttcaaa 1980
gaaaaaggac gcattgcaga tcttcgtgaa gttgtggatg ccaatgtaga agcgaaagcc 2040
ggaattgggc atactcgctg ggcgacacac ggcgaaccaa gctatctgaa cgctcacccg 2100
catcaaagcg cactgggccg ctttacactt gttcacaacg gcgtgatcga gaactatgtt 2160
cagctgaagc aagagtattt gcaagatgta gagctcaaaa gtgacaccga tacagaagta 2220
gtcgttcaag taatcgagca attcgtcaat ggaggacttg agacagaaga agcgttccgc 2280
aaaacactta cactgttaaa aggctcttat gcaattgctt tattcgataa cgacaacaga 2340
gaaacgattt ttgtagcgaa aaacaaaagc cctctattag taggtcttgg agatacattc 2400
aacgtcgtag catctgatgc gatggcgatg cttcaagtaa ccaacgaata cgtagagctg 2460
atggataaag aaatggttat cgtcactgat gaccaagttg tcatcaaaaa ccttgatggt 2520
gacgtgatta cacgtgcgtc ttatattgct gagcttgatg ccagtgatat cgaaaaaggc 2580
acgtaccctc actacatgtt gaaagaaacg gatgagcagc ctgttgttat gcgcaaaatc 2640
atccaaacgt atcaagatga aaacggcaag ctgtctgtgc ctggcgatat cgctgccgct 2700
gtagcggaag cggaccgcat ctatatcatt ggctgcggaa caagctacca tgcaggactt 2760
gtcggtaaac aatatattga aatgtgggca aacgtgccgg ttgaagtgca tgtagcgagt 2820
gaattctcct acaacatgcc gcttctgtct aagaaaccgc tcttcatttt cctttctcaa 2880
agcggagaaa cagcagacag ccgcgcggta ctcgttcaag tcaaagcgct cggacacaaa 2940
gccctgacaa tcacaaacgt acctggatca acgctttctc gtgaagctga ctatacattg 3000
ctgcttcatg caggccctga gatcgctgtt gcgtcaacga aagcatacac tgcacaaatc 3060
gcagttctgg cggttcttgc ttctgtggct gctgacaaaa atggcatcaa tatcggattt 3120
gacctcgtca aagaactcgg tatcgctgca aacgcaatgg aagctctatg cgaccagaaa 3180
gacgaaatgg aaatgatcgc tcgtgaatac ctgactgtat ccagaaatgc tttcttcatc 3240
ggacgcggcc ttgactactt cgtatgtgtc gaaggcgcac tgaagctgaa agagatttct 3300
tacatccagg cagaaggttt tgccggcggt gagctaaagc acggaacgat tgccttgatc 3360
gaacaaggaa caccagtatt cgcactggca actcaagagc atgtaaacct aagcatccgc 3420
ggaaacgtca aagaagttgc tgctcgcgga gcaaacacat gcatcatctc actgaaaggc 3480
ctagacgatg cggatgacag attcgtattg ccggaagtaa acccagcgct tgctccgttg 3540
gtatctgttg ttccattgca gctgatcgct tactatgctg cactgcatcg cggctgtgat 3600
gtggataaac ctcgtaacct tgcgaagagt gttactgtgg agtaaaattg tgtaaatgaa 3660
attgattttt tgttgtgctc aggttaagat ttaatttgat gtgttaatga gaatgttggg 3720
aatagactga tttttttgag cgtgctgcat aggaggttga aatgcgaaaa acgttttttt 3780
cgaagatttc atttatgctg attgccattt tattgatgtg gctgaaaacg tatgctgttt 3840
acaaaaccag ttttcatatt aaaatcgaca atctaacaca ggaatttatt ctgtttatca 3900
acccattgag ttttttgttg cttatttttg gcctcagcct gtttttaaaa ggcaaaaaca 3960
gaaatcgcta cattatcgcg atgagctgtc ttgtcacgtt tgtattgctg gcaaatatgg 4020
ttttttaccg tttttacaat gatttcttaa caatccctgt tctttttcaa acgagcaata 4080
tgggtgatct cggaagcagc atcggaacac ttcttgagcc gacagacctc ctattagctg 4140
tagatattgc ggttttaata tggcttcaca tccggcaaaa agcttttcaa tcggacattc 4200
cctcaacgaa aaatgaacgg gcggcttatt ttttgttcgt tgcttctgtt tatttcttca 4260
acctgggctt gtctgaggcg gaaagacctc agctattgac acgctcattt gacagagaaa 4320
tgcttgttaa aaacattagc ctgtttaatt ttcatattta cgatggcgtt cttcagtcaa 4380
agcaatccgc acagagagcg ttggcagaca gcaacagcct gacggagatt gaaaactacg 4440
taaccgctaa tgcgaaggat gccaacaaac gcttattcgg cgctgcaaaa ggaaggaacg 4500
tcattctcgt atccttagag tcgacgcaaa gcttcgtgat taatgaaaaa ttgaatggag 4560
aagaaatcac gccttttctg aatgacttta taaaacagag ctacaacttt aataatgttt 4620
accaccaaac aggccagggg aaaac 4645
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 6- phosphorylated amino glucose synzyme（GlmS）, obtain recombinant bacterium BSGNKS；The recombined bacillus subtilis BSGNK is
With 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δs of B. subtilis
glck ::Lox72 is host, controls the recombinant expression of glms, GNA1 with promoter PxylA, P43 respectively.
2. recombined bacillus subtilis as described in claim 1, it is characterised in that：The 6- phosphorylated amino glucoses synzyme
（GlmS）GeneID in encoding gene glmS such as NCBI:Shown in 938736.
3. recombined bacillus subtilis as described in claim 1, it is characterised in that：The 6- phosphorylated amino glucoses synzyme
（GlmS）Structure integrant expression frame be SEQID NO.1, and be integrated into the sites glcK through homologous recombination.
4. being overexpressed 6- phosphorylated amino glucose synzyme using recombined bacillus subtilis as claimed in claim 1,2 or 3
Promote acetylglucosamine synthetic method, it is characterised in that：Using recombined bacillus subtilis BSGNK as starting strain,
It is overexpressed 6- phosphorylated amino glucose synzyme（GlmS）, the recombinant bacterium BSGNKS of gained for producing acetylglucosamine,
The recombined bacillus subtilis BSGNK is with 168 Δ nagP Δ gamP Δ gamA Δs nagA of B. subtilis
Δ nagB Δ ldh Δ pta Δ glck ::Lox72 is host, respectively with promoter PxylA、P43Control glms, GNA1
Recombinant expression.
5. recombined bacillus subtilis as claimed in claim 4, which is overexpressed 6- phosphorylated amino glucose synzyme, promotes acetyl ammonia
Base glucose synthetic method, it is characterised in that：Utilize the recombinant bacterium BSGNKS combinations complex medium laminating production acetyl
Glucosamine.
6. recombined bacillus subtilis as claimed in claim 5, which is overexpressed 6- phosphorylated amino glucose synzyme, promotes acetyl ammonia
Base glucose synthetic method, 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 6- phosphorylated amino glucose synzyme, promotes acetyl ammonia
Base glucose synthetic method, it is characterised in that：Second is produced using the recombinant bacterium BSGNKS combining with fermentation culture medium fermentation method
Acylamino- glucose.
8. recombined bacillus subtilis as claimed in claim 7, which is overexpressed 6- phosphorylated amino glucose synzyme, promotes acetyl ammonia
Base glucose synthetic method, it is characterised in that：By the seed culture fluid of the recombined bacillus subtilis after activation so that
Few 10% inoculum concentration, which is transferred in the fermentation medium, to be 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.
9. recombined bacillus subtilis as claimed in claim 8, which is overexpressed 6- phosphorylated amino glucose synzyme, promotes acetyl ammonia
Base glucose synthetic method, 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 6- phosphorylated amino glucose synzyme, promotes acetyl
Glucosamine synthetic method, it is characterised in that：The derivant includes xylose, and the dosage of the xylose is 5 g/L；It is described
The liquid amount of shaking flask is 50 mL.
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| 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 |
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