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CN106967659A - A kind of structure and fermentation process of the antibiotic-free resistance recombined bacillus subtilis for expressing glutamate decarboxylase - Google Patents

A kind of structure and fermentation process of the antibiotic-free resistance recombined bacillus subtilis for expressing glutamate decarboxylase Download PDF

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CN106967659A
CN106967659A CN201710193236.5A CN201710193236A CN106967659A CN 106967659 A CN106967659 A CN 106967659A CN 201710193236 A CN201710193236 A CN 201710193236A CN 106967659 A CN106967659 A CN 106967659A
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dal
bacillus subtilis
gad
hpaii
gene
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江波
沐万孟
丁汪洋
张涛
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Jiangnan University
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    • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)

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Abstract

A kind of structure and fermentation process of the antibiotic-free resistance recombined bacillus subtilis for expressing glutamate decarboxylase, belong to technical field of bioengineering.Using bacillus subtilis WB600 as starting strain, knock out D alanine racemases enzyme gene on its chromosome (dal) obtain D alanine deficiencies WB600 (dal ‑)；Using overlap extension pcr by after optimizationgadGene and antibiotic-free resistance expression vector pUB110（Its antibiotics resistance gene is replaced by D alanine racemase enzyme genes）Fusion obtains polymer, then by polymer be transformed into bacillus subtilis WB600 (dal ‑) competence in, polymer is recombinated in host, is obtained recombinant plasmid pUB HpaII P43 gad dal, is named as bacillus subtilis（Bacillus subtilis）SK44.001, deposit number CCTCC NO：M 2016774.Its zymotic fluid enzyme activity is up to 8.6U/mL, with important industrial application value.

Description

A kind of antibiotic-free resistance recombined bacillus subtilis for expressing glutamate decarboxylase Build and fermentation process
Technical field
The present invention relates to it is a kind of express glutamate decarboxylase antibiotic-free resistance recombined bacillus subtilis structure and Fermentation process, belongs to technical field of bioengineering.
Background technology
GABA (GABA) is a kind of naturally occurring nonprotein amino acid, is widely distributed in animal and plant body In, research is found, GABA, which has, improves cardiovascular, treats sacred disease, improves hepatic and renal function and regulation endocrine The important function such as system, therefore it has broad application prospects in terms of drug therapy and functional food.
Preparing the method for GABA mainly has chemical method, animals and plants concentration method and Microbe synthesis method three major types side Method.Chemical synthesis has used poisonous chemical reagent in production, and food can not be added to by producing obtained GABA In product；And GABA content in animals and plants is very low, it is faced with by plants enriched method to obtain GABA The problem of cost is too high；Microbe synthesis method refers to utilize the glutamic acid decarboxylase enzymes biocatalysis L- paddy contained in microbial cell Propylhomoserin sodium (L-MSG) occurs decarboxylic reaction and prepares GABA.
Using the wild mushroom fermenting and producing GABA such as lactic acid bacteria be faced with cultivation cycle length, producing enzyme efficiency it is low, point The shortcomings of complicated from purification step；Although can be greatly improved with Bacillus coli expression external source glutamate decarboxylase enzyme activity, due to Escherichia coli are Gram-negative bacterias, and endotoxin can be produced during the fermentation, so being production bacterial strain life using Escherichia coli Production GABA cannot be used for food industry.Bacillus subtilis has the long-term history for preparing fermented food, by the U.S. The department such as FDA and the Ministry of Agriculture is approved as grade-safe bacterial strain, it has, and culture is simple and quick, protein secretion ability is strong, The features such as albumen of expression is difficult to form inclusion body, non-pathogenic, is the preferable expressive host for producing various industrial enzymes at present.
The content of the invention
The present invention provides the recombined bacillus subtilis of the grade-safe of one plant of expression glutamate decarboxylase, and can be used for Conversion sodium glutamate prepares GABA, has great importance to the GABA for producing food-grade.
Technical scheme is as follows：
The antibiotic-free resistance recombined bacillus subtilis of one plant of expression glutamate decarboxylase, its Classification And Nomenclature is bacillus subtilis Bacterium（Bacillus subtilis）SK44.001, has been preserved in China typical culture collection center, deposit number CCTCC NO：M 2016774, its be by recombinant plasmid pUB-HpaII-P43-gad-dal import host cell WB600 (dal -) and obtain , consisting of WB600/pUB-HpaII-P43-gad-dal, it is capable of synthetic amino acid array SEQ ID No.1 paddy ammonia Acid decarboxylase.
A kind of glutamate decarboxylase, its amino acid sequence is as shown in SEQ ID No.1.
The encoding gene of described glutamate decarboxylase is with the Lactococcus lactis after codon optimizationLactococcus lactis subsp. lactisIL1403'sgadWhat gene cloning was obtained, it is originalgadNucleotide sequence is such as Shown in SEQ ID No.2, the gad nucleotide sequences after optimization are as shown in SEQ ID No.3
A kind of vector plasmid pUB-HpaII-P43-dal, its nucleotide sequence is SEQ ID No.4.
A kind of recombinant plasmid pUB-HpaII-P43-gad-dal, contains the gad nucleic acid sequence SEQs ID after the optimization No.3 and pUB-HpaII-P43-dal more control sequences SEQ ID No.4.
The construction method of the antibiotic-free resistance recombined bacillus subtilis of described expression glutamate decarboxylase, including will Lactococcus lactis after codon optimizationLactococcus lactis subsp. lactisIL1403'sgadBase Because of sequence (the nucleotide sequence such as SEQ ID No.3 after its optimization) and double promoter expression vector pUB-HpaII-P43-dal (its Nucleotide sequence such as SEQ ID No.4) merged by PCR modes and obtain plasmid pUB-HpaII-P43-gad-dal；And turn plasmid Change to the selection markers of deficiency containing D-alanine bacillus subtilis WB600 (dal -) middle expression, obtain one plant of expression paddy ammonia The antibiotic-free resistance recombined bacillus subtilis WB600/pUB-HpaII-P43-gad-dal of acid decarboxylase, is named as withered grass Bacillus (Bacillus subtilis) SK 44.001, deposit number CCTCC NO：M 2016774.
Concretely comprise the following steps：
（1）Host cell WB600 (dal -) structure：WB600(dal -) be D-alanine racemase gene defect withered grass bud Spore bacillus,
（a）D-alanine racemase gene on bacillus subtilis WB600 chromosomesdalUpstream and downstream respectively choose 900bp The fragment of left and right length is expanded two sections of homology arms by PCR as homology arm, and glue reclaim；Chooselox71-spc-lox66 Resistance gene fragment as selection markers, by upstream and downstream homology arm andlox71-spc-lox66 fragments are by fusion DNA vaccine by three sections Gene links together；PCR primer is transferred in bacillus subtilis WB600 competence, is containing spectinomycinspcFlat board Upper screening；Due to the presence of homology arm, occur homologous recombination, knock out the D-alanine on bacillus subtilis WB600 chromosomes Racemase genedal, obtain D-alanine deficiency host WB600 (dal -)-Spc。
（b）PTSC (Bacillus Genetic Stock Center accession no. ECE204) plasmid is imported Above-mentioned host WB600 (dal -)-SpcIn, screened on the flat board containing erythromycin, at IPTG (isopropylthiogalactoside) Induction under, express recombinase Cre, in the presence of recombinase Cre,lox71 Heslox66 sites are recombinated, willspcResistance Gene elmination, and obtain one plant of D-alanine deficiency bacillus subtilis WB600 (dal -)。
（2）Using P7 and P8 as primer, using plasmid pET-22b-gad as template, PCR amplifications obtain linear fragment 1.
（3）Using P9 and P10 as primer, using plasmid pUB-HpaII-P43-gad-dal as template, PCR amplifications obtain linear Fragment 2.
（4）With linear fragment 1 and linear fragment 2 primer each other, PCR amplifications obtain polymer fragment.
（5）Using two steps (GM I, GM II) method prepare antibiotic-free resistance bacillus subtilis WB600 (dal -) sense By state cell.
（6）By step（4）Polymer fragment be transformed into step（5）In obtained competent cell, be coated on without Antibiotic screens without the LB flat boards of alanine and obtains recombinant bacterial strain WB600/pUB110-P43-HpaII-gad-dal.
The fermentation process of recombined bacillus subtilis (WB600/pUB-HpaII-P43-gad-dal) is as follows：
Seed liquor culture：Single bacterium colony on picking flat board, is inoculated in 4 mL LB Tube propagation bases, 37 DEG C, 200 r/min trainings Support 12 h.The LB culture medium prescriptions are：The g/L of tryptone 10；The g/L of yeast extract 5；The g/L of sodium chloride 10；Using Deionized water is prepared, and sterilising conditions are 121 DEG C, 20 min.
Fermented and cultured：Seed liquor is transferred in 50 mL fermentation mediums by volume fraction 2%, 37 DEG C, 200 r/min trainings Final zymotic fluid is obtained after supporting 20h.The fermentative medium formula is：The g/L of soy peptone 25；The g/L of lactose 5； Na2HPO4·12H2O 3g/L；MnSO4·H2O 0.1g/L；Prepared using deionized water, sterilising conditions are 115 DEG C, 30min.
Described recombined bacillus subtilis WB600/pUB-HpaII-P43-gad-dal application, the glutamic acid of expression Decarboxylase can be catalyzed sodium glutamate or glutamic acid production GABA（GABA）, can be in chemistry, food and pharmaceutical field Middle application.
Beneficial effects of the present invention：The present invention constructs a kind of antibiotic-free for being capable of high efficient expression glutamate decarboxylase and resisted Property recombined bacillus subtilis, make its under specific fermentation medium condition of culture can high efficient expression glutamate decarboxylase, Constructed engineered strain protein expression ability is strong, and the threat without contaminated with endotoxins is safe and reliable, and the enzyme activity of zymotic fluid is most High reachable 8.6U/mL.Glutamate decarboxylase obtained by the present invention, can be using L-sodium as substrate, in gentle condition GABA is obtained by bioconversion down, technique is simple, environment-friendly, the recombinant bacterium is in chemistry, food and pharmacy neck There is important application value in domain.
Biological material specimens preservation：The antibiotic-free resistance recombined bacillus subtilis of one plant of expression glutamate decarboxylase, It has been preserved in China typical culture collection center, abbreviation CCTCC, address is：Wuhan, China, Wuhan University, preservation date On December 21st, 2016, Classification And Nomenclature is bacillus subtilis SK44.001（Bacillus subtilis）SK44.001, preservation Numbering is CCTCC NO：M 2016774.
Brief description of the drawings
Fig. 1：The nucleic acid electrophoresis figure for the product that PCR is obtained.M、DL 5000 DNA Marker；1st, linear fragment 1；2nd, it is linear Fragment 2；3rd, polymer fragment.
Fig. 2：Recombinant plasmid pUB-HpaII-P43-gad-dal structure collection of illustrative plates.
Fig. 3：Recombined bacillus subtilis WB600/pUB-HpaII-P43-gad-dal growth curve and enzyme activity curve.
Embodiment
Come that the present invention is furture elucidated by the following examples, the following example for illustration purposes not for limitation this Invention scope.
Embodiment 1：D-alanine deficiency bacillus subtilis WB600 (dal - ) structure
Using plasmid p7S6 as template, primer pair P3/P4 willlox71-spc-lox66 antibiotics resistance gene fragment amplifications are simultaneously returned Receive.D-alanine racemase gene on bacillus subtilis WB600 chromosomesdalBoth sides selection 800-900bp length The homology region at two ends is expanded and reclaimed with primer pair P1/P2 and P5/P6 respectively as homology region by fragment.Use primer To P1/P6 by two ends homology arm fragment and antibioticlox71-spc-lox66 are merged by round pcr.
PCR reaction systems：Following reagent is sequentially added into 0.2mL PCR pipes：Each 1.5 μ L of upstream and downstream primer；5μL Phusion HF buffer (5×)；2μL 10mM dNTP mix (2.5mM each)；2 μ L upstreams homology arms；0.5μLlox71-spc-lox66；2 μ L downstreams homology arms；0.5 μ L Phusion high-fidelity archaeal dna polymerases；Plus distilled water To the μ L of final volume 50.
PCR amplification conditions：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 30s, 55 DEG C of annealing 15s, 72 DEG C of extension 1min（30 are followed Ring）；72 DEG C of extension 10min.
PCR primer is converted to bacillus subtilis WB600 competence, is containing antibioticspcWith putting down for D-alanine Screened on plate, obtain one plant of WB600 (dal - )- Spc。
Import pTSC plasmids（Agricultural University Of Nanjing, doctor Yan Xin give, NCBI accession no.EU864234）, Cre recombinases, in the presence of recombinase, lox are expressed under IPTG induction71And lox66Site is recombinated, resistant genespcIt is deleted.
It is final obtain D-alanine deficiency bacillus subtilis WB600 (dal - )。
Embodiment 2：Grade-safe plasmid pUB-HpaII-P43-gad-dal structure
Material and reagent：Archaeal dna polymerase PrimeSTAR Max DNA Polymerase (2 ×) and the DNA Marker of DL 5000 Purchased from the precious bioengineering Co., Ltd in Dalian；The a small amount of extraction agent boxes of SanPrep pillar DNAs and SanPrep pillar DNA glue QIAquick Gel Extraction Kit is purchased from Sangon Biotech (Shanghai) Co., Ltd.；Primer is limited by giving birth to work bioengineering (Shanghai) share Company synthesizes；Plasmid pET-22b-gad is synthesized by Shanghai Jierui Biology Engineering Co., Ltd；Other common agents are purchased from traditional Chinese medicines Chemical reagent Co., Ltd of group.
With P7, P8 is primer, and pET-22b-gad is template, and PCR expands gad genes,
Reaction system：
pET-22b-gad 1μL
P7 1.25μL
P8 1.25μL
ddH2O 21.5μL
PrimeSTAR Max DNA Polymerase(2×) 25μL
Reaction condition：98 DEG C of min of pre-degeneration 2；98 DEG C of 10 s that unwind, 55 DEG C of annealing 5 s, 72 DEG C of extension 30s, totally 30 circulations； 72 DEG C of 5 min of extension.
With P9, P10 is primer, and pUB-HpaII-P43-dal are template, PCR amplification pUB-HpaII-P43-dal genes (have compared with template withgadThe fragment of gene overlap),
Reaction system：
pUB-HpaII-P43 -dal 1μL
P8 1.25μL
P9 1.25μL
ddH2O 21.5μL
PrimeSTAR Max DNA Polymerase(2×) 25μL
Reaction condition：98 DEG C of min of pre-degeneration 2；98 DEG C of 10 s that unwind, 55 DEG C of annealing 5 s, 72 DEG C of extension s of 1 min 20, totally 30 Individual circulation；72 DEG C of 5 min of extension.
Glue reclaim purified pcr product, the concentration of two linear fragments is determined with NanoDrop, according to the ︰ 1 of mol ratio 1 each other Masterplate PCR, obtains polymer fragment.
Reaction system：
gad 1μL
pUB-HpaII-P43 -dal 1μL
ddH2O 23μL
PrimeSTAR Max DNA Polymerase(2×) 25μL
Reaction condition：98 DEG C of min of pre-degeneration 2；98 DEG C of 10 s that unwind, 60 DEG C of annealing 5 s, 72 DEG C of extension s of 1 min 50, totally 30 Individual circulation；72 DEG C of 5 min of extension.
By polymer fragment import bacillus subtilis WB600 (dal -) competent cell, cultivated on LB flat boards, screening Obtain engineering bacteria WB600/pUB-HpaII-P43-gad-dal.
The fermentation expression glutamate decarboxylase of the recombined bacillus subtilis of embodiment 3
Picking single bacterium colony is inoculated in 4 mL LB culture mediums on flat board, 37 DEG C, and 200 r/min cultivate 12 h；According to volume integral Seed liquor is accessed 50 mL fermentation mediums by the inoculum concentration of number 2%, and 37 DEG C, 200 r/min are cultivated to 21h, and timing sampling surveys hair The OD600 of zymotic fluid and enzyme activity (Fig. 3).
Involved culture medium prescription：
LB culture mediums：The g/L of tryptone 10；The g/L of yeast extract 5；The g/L of sodium chloride 10；Prepared using deionized water, Sterilising conditions are 121 DEG C, 20 min.
Fermentation medium：The g/L of soy peptone 25；The g/L of lactose 5；Na2HPO4·12H2O 3g/L；MnSO4·H2O 0.1g/L；Prepared using deionized water, sterilising conditions are 115 DEG C, 30min.
Enzyme activity of fermenting definition：50 DEG C, under pH 4.5 reaction condition, needed for 1 μm of ol GABA of generation per minute Enzyme amount be defined as 1 enzyme-activity unit (U/mL).
Ferment enzyme activity assay method：The mL of cumulative volume 1 is reacted, including：PH 4.5,0.1 mol/L acetate salt buffers Liquid, 0.1 mol/L sodium glutamates, the thalline that 1mL zymotic fluids are centrifuged.
Reaction condition：Temperature 50 C, the min of time 10.
Reaction terminates the rear min of boiling water boiling 10 and gone out enzyme, using HPLC detections after centrifuging and taking supernatant.
SEQ ID No.1
MET Leu Tyr Gly Lys Glu Asn Arg Asp Glu Ala Glu Phe Leu Glu
5 10 15
Pro Ile Phe Gly Ser Glu Ser Glu Gln Val Asp Leu Pro Lys Tyr
20 25 30
Lys Leu Ala Gln Gln Ser Ile Glu Pro Arg Val Ala Tyr Gln Leu
35 40 45
Val Gln Asp Glu MET Leu Asp Glu Gly Asn Ala Arg Leu Asn Leu
50 55 60
Ala Thr Phe Cys Gln Thr Tyr MET Glu Pro Glu Ala Val Lys Leu
65 70 75
MET Ser Gln Thr Leu Glu Lys Asn Ala Ile Asp Lys Ser Glu Tyr
80 85 90
Pro Arg Thr Thr Glu Ile Glu Asn Arg Cys Val Asn MET Ile Ala
95 100 105
Asp Leu Trp Asn Ala Ser Glu Lys Glu Lys Phe MET Gly Thr Ser
110 115 120
Thr Ile Gly Ser Ser Glu Ala Cys MET Leu Gly Gly MET Ala MET
125 130 135
Lys Phe Ser Trp Arg Lys Arg Ala Glu Lys Leu Gly Leu Asp Ile
140 145 150
Asn Ala Lys Lys Pro Asn Leu Val Ile Ser Ser Gly Tyr Gln Val
155 160 165
Cys Trp Glu Lys Phe Cys Ile Tyr Trp Asp Ile Glu MET Arg Glu
170 175 180
Val Pro MET Asp Lys Glu His MET Ser Ile Asn Leu Asp Lys Val
185 190 195
MET Asp Tyr Val Asp Glu Tyr Thr Ile Gly Val Val Gly Ile MET
200 205 210
Gly Ile Thr Tyr Thr Gly Arg Tyr Asp Asp Ile Lys Ala Leu Asp
215 220 225
Asn Leu Ile Glu Glu Tyr Asn Lys Gln Thr Asp Tyr Lys Val Tyr
230 235 240
Ile His Val Asp Ala Ala Ser Gly Gly Leu Tyr Ala Pro Phe Val
245 250 255
Glu Pro Glu Leu Glu Trp Asp Phe Arg Leu Lys Asn Val Ile Ser
260 265 270
Ile Asn Thr Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val
275 280 285
Gly Trp Val Leu Trp Arg Asp Lys Lys Tyr Leu Pro Glu Glu Leu
290 295 300
Ile Phe Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr MET Ala
305 310 315
Ile Asn Phe Ser His Ser Ala Ser Gln Leu Ile Gly Gln Tyr Tyr
320 325 330
Asn Phe Val Arg Tyr Gly Phe Asp Gly Tyr Lys Ala Ile His Glu
335 340 345
Arg Thr His Lys Val Ala MET Phe Leu Ala Lys Glu Ile Glu Lys
350 355 360
Thr Gly MET Phe Glu Ile MET Asn Asp Gly Ser Gln Leu Pro Ile
365 370 375
Val Cys Tyr Lys Leu Lys Glu Asp Ser Asn Arg Gly Trp Asn Leu
380 385 390
Tyr Asp Leu Ala Asp Arg Leu Leu MET Lys Gly Trp Gln Val Pro
395 400 405
Ala Tyr Pro Leu Pro Lys Asn Leu Glu Asn Glu Ile Ile Gln Arg
410 415 420
Leu Val Ile Arg Ala Asp Phe Gly MET Asn MET Ala Phe Asn Tyr
425 430 435
Val Gln Asp MET Gln Glu Ala Ile Glu Ala Leu Asn Lys Ala His
440 445 450
Ile Leu Tyr His Glu Glu Pro Glu Asn Lys Thr Tyr Gly Phe Thr
455 460 465
His***
466
SEQ ID No.2
atgttatacg gaaaagaaaa tcgcgatgaa gcagagttct tggaaccaat ttttggttca 60
gaaagtgaac aagtggattt gcctaaatat aaattagctc aacaatcaat tgaacctcga 120
gtggcctatc agttagttca agatgaaatg ttagatgaag ggaacgctcg tttaaattta 180
gccacattct gtcaaactta tatggaacct gaagcagtca aactaatgag tcaaaccttg 240
gaaaaaaatg caattgataa atcggaatat ccaagaacaa ctgaaattga gaaccgttgc 300
gtcaacatga tcgctgacct ttggaatgcg agtgaaaaag aaaaatttat ggggacttca 360
acgattggtt cttcagaagc ttgtatgctt ggtggaatgg ccatgaagtt ttcttggcgc 420
aagcgagcag aaaaattggg attagatatt aatgcgaaaa agccaaactt agttatttca 480
tctggttatc aagtttgctg ggaaaaattc tgtatttatt gggatattga aatgcgagaa 540
gtgccaatgg ataaagaaca tatgtcaatc aatttggaca aggtgatgga ttatgttgat 600
gagtacacga ttggtgtagt tggtattatg gggattactt atactggtcg ttatgatgat 660
atcaaagctt tggataattt aattgaagaa tataataaac agacagacta taaagtttat 720
attcacgtag atgctgcttc aggaggactt tatgctccct ttgttgagcc agaacttgag 780
tgggatttcc gtttgaaaaa tgtcatttca atcaatacct caggccataa atatggttta 840
gtttatcctg gtgtaggttg ggttttgtgg cgtgacaaaa aatatttacc agaagaatta 900
atttttaaag taagttatct tggaggagaa ctaccaacga tggccattaa tttttctcat 960
agtgcctctc aattaattgg tcaatattat aattttgtac gttatggatt tgatggatat 1020
aaagctattc atgagagaac acataaagta gccatgtttt tagcaaaaga aattgaaaaa 1080
actggaatgt ttgaaattat gaacgatggg tcacaattgc caattgtctg ctataaatta 1140
aaagaagatt caaatcgagg ttggaatctt tatgatttgg cggaccgttt attaatgaag 1200
ggatggcaag tgcctgctta tccacttccc aaaaatttgg aaaatgaaat cattcaacgt 1260
ttagtgattc gagcagattt tgggatgaat atggcattta actatgttca agatatgcaa 1320
gaagcaattg aggctttaaa taaggctcat attctatatc atgaagagcc tgaaaataaa 1380
acatatggat ttactcacta a 1401
SEQ ID No.3
atgttgtatg gcaaagaaaa tagagatgaa gcggaatttc tggaaccgat ctttggcagc 60
gaatcagaac aagtcgatct tcctaaatac aaacttgcac aacagagcat tgaacctcgg 120
gtcgcttatc agttagtcca ggatgaaatg ttagatgaag gcaatgcacg ccttaatctt 180
gcgacgtttt gccagacgta tatggaacct gaagcagtca aactgatgtc tcagacactt 240
gagaagaacg ctattgataa atctgaatat cctcgcacga cggaaatcga aaatcggtgc 300
gtcaatatga ttgcggatct gtggaatgcg agcgaaaaag aaaaattcat gggcacgtct 360
acaatcggct ctagcgaagc gtgtatgctt ggcggcatgg cgatgaaatt cagctggcgc 420
aaacgcgcgg aaaaactggg actggatatt aatgctaaaa aacctaatct tgtaatcagc 480
tcaggctatc aggtgtgttg ggaaaaattt tgcatctatt gggacattga aatgcgcgaa 540
gttccgatgg ataaagaaca catgagcatc aatcttgata aagtcatgga ttatgtcgat 600
gaatatacga tcggcgtggt cggcatcatg ggcataacct atacgggccg gtatgatgat 660
attaaagcgt tagataatct tattgaagaa tataataagc agacggatta caaagtgtat 720
atccatgtcg atgctgcttc cggaggcctt tatgctccgt ttgtcgaacc ggaattagaa 780
tgggattttc gtcttaaaaa tgtcatctct attaatacga gcggccataa atatggactt 840
gtgtatccgg gagtgggctg ggtcctgtgg agagataaaa aatatctgcc ggaagaactt 900
atctttaaag tgagctatct cggtggagaa ctgccgacaa tggcgatcaa tttttcacat 960
tcagcttcac aactgattgg acagtattat aattttgttc gctatggctt tgatggctac 1020
aaagcgatcc atgaacgtac acataaagtt gcaatgtttc ttgctaaaga aatcgaaaaa 1080
acgggcatgt ttgaaatcat gaatgatggc tcacagttac cgatcgtgtg ctacaaactt 1140
aaagaagatt ctaatagagg ctggaatctg tatgatctag cagatcggtt actgatgaaa 1200
ggctggcagg tccctgcgta tccgctgcct aaaaacctgg aaaatgagat catccaacgc 1260
ctggtcattc gcgcagattt tggcatgaat atggcgttta attatgtcca agatatgcag 1320
gaagcaatcg aagcacttaa caaagctcat attctgtatc atgaagaacc ggaaaacaaa 1380
acgtatggct ttacacatta a 1401
SEQ ID No.4
gcagacatgc aaggaattcc ttaaggaaag cccgaaacaa tcgtcggcct agagtcacca 60
ccaccaccac caccacatgt aaggagagaa tggatattac catggcgata gtgaaatata 120
aaatgtatta gcgcgcgaaa aaaagtgcgg gtaaagattt ttacatttta tttacattct 180
taaaagtcgg taatgtcgaa accgtttttt tattcatttg gttactatgt gctttagtgc 240
cgtttttgcg tttgtcgggg ccgccgtcgc aggaaccggc gaaatcgttc tcccactaca 300
aacagtcaat aatttagttg gcatacaagt taccgacata aattttcaag ttcgcttttg 360
tatggtggat agtaatacta aagtgaaaaa cgtaagatgt ttgacgtatt gagtatacat 420
ttagcgagga aaaatccacc gtgtttacac tccgtaaaag cgagaaaggc cgttggtgaa 480
ggttcatttc atattgtgtg atatgaaata taagtatttc acacacgaga cgctccgaca 540
gccgtcacgg ctggttttgg tattttggaa attctggaaa gaaaaaaaat gctctttttt 600
ctttgttttt ttggacggga gacggtggag tcgtttcccc ccaaaacgag agcacgagca 660
aatttttagt cgttccctgt ccatcataaa aaactcttct agtgagtttt ttagaggtgg 720
aaatttggga acggttaaaa ataaaacagg caaaacagat cgaatggctt tcggtctgag 780
tcgttcttat tttaaaaata acagaaagcc aaaagatcac attgcctgtt ttggtgagtt 840
ttattttttc tatgttctct ccagagagca tagaaaataa gtcgttagcg cgggctaacg 900
acttgtctaa ttattatcta acttaagagg taaatccatt caattaacga atataaatgg 960
acgttatttc ctaaagaatg aaggtaatat gagggtaaaa ggtttttgta tgccccttgt 1020
gcccttgaat aacatgtccg gtggagtatc aattaccaaa gctcggaagg acgttagagt 1080
aggtaccttt atataagtag ggggacggcc ggataattac actgaaaaca cgggccgcct 1140
ataaggacta ggtcgaggtg gtatttaacc aggtacgttt aagccggccg ttaaaagtcc 1200
gcaaaaggga agtgttccta cagccaggga aagttaaaag cctcggtcgg caggcgtatc 1260
ggatgtccgt ggcagggcta ggtacacaga aaaaggcgac acatgagccg aggcatcgac 1320
tgcgagagcg gaaaagacta gtcaaactgt acactgtcac agcttacgtc ccatttacgg 1380
cctgcgtcga ctttgccata gagcaggctg tacagtcgtc tgcccgcttc cggtatgtac 1440
ggctacggct tagactgacg taattttttc ggaaaaaagt cggcctcagg tcgccgcgac 1500
aagcgcgtca cctggtaatc taagaaattg ccgtcgcctc gttagtcgag aaatttcgcg 1560
agtttgacgt aattctttat cggagaaaga aaaagtaggc gacagcgttt tacccattta 1620
tggggaaacg tgaaatttgc tcccaacgcc agttcttaac ggtagtgcaa gacttgaaga 1680
aggagacaaa aatgtggttc agacaagtag gggcatagct ggaagtctac ttttacttct 1740
cttggaaaaa agcacaccgc ccgacggagg acttcggtaa gttgtcttat tggacaattc 1800
cagtgcagta tgagtcgtcg ctaacggtgt atgaggcccc cttggcgcgg ttcgtggtta 1860
tatccgcgga agttagggaa aaacgcgtca ctttagcgaa gtaggtttta ccggtgccgg 1920
ttcgtacttc gtggacgcag ttctcgtcgg aaacgacaaa gacgtagtgg tacgggcatc 1980
cgcaaacgaa agtgttgacg gtagttcacc tgtacaagtg gctatacaaa aaagtataac 2040
gactgtaaaa ggaaatagcg cctgttcagt taaaggcggg tgcatagaga catttttcca 2100
aaacacgagt aatgctgtga aggatcgaaa ataagttata gtaaatgtat agtatgattt 2160
taatttccga tttccctttg ctacagattg ctttttttcc ggtttagtac aaaccggaaa 2220
ccgccaataa agctactaac agggacgtac gaaaagcgat gacagtggta aaggagaaat 2280
ctttctagaa gggaaaagtc tattaaaatc taaacgaaaa gatttattct tataaacctc 2340
tcgtggcaag aataagtcga taattattga gcagaaggat tcgtaggaag ttaggaaaat 2400
tattgttaat atcgtagatt agaagttgtt tgaccgggca aacaacttga tgagaaatta 2460
ttttattaaa aaggcaaggg ttaaggtgta acgttattat cttttaggta gaagtagccg 2520
aaaaagcagt agtagacata cttagtttag cggaagaaga cacagtagtt ccaaattaaa 2580
aaatacataa agaaaattgt ttggtggtat cctctaattg gaaaatgcca catttggaag 2640
gaggtttagt ctgtttgcaa agtttaagaa aagaagtagt agccagtatt ttaggcatag 2700
gaaatgtcct ataaaacgtc aaagcagtta acggctaaca tataggctaa atataaataa 2760
aaagccagct tagtaaactt gaaaatgtaa acctagtatc agattaaagt aacggaaaaa 2820
ggttttaact taggtaacaa aaactaagtg catcaaaaga cataagaatt ttattcaacc 2880
aaggtgtgta tggttatgta cgtacacgac taatattctt aatagaaata ataaataaca 2940
gtgaaggcaa cgtgcgtatt ttggttgttc taaaaataat taaaaaaata taacgtagta 3000
agccgcttta ggaactcggt atagactgtt tgagaataaa ttaagaagcg gtagtatttg 3060
taaaaattga caattacact ctttgttggt tgcttgacaa ccgaaaacaa attattgaag 3120
tcgttgttgg aaaacactga cttacggtac aaagtaacga gaggaggtca acgtgtaacc 3180
tgtttcggac ctaaatgttt tggtgtgagc tatgttgaaa gaaagcggac aaagtgctaa 3240
aacaaatatg agattataaa gtcgtgttag aaaatgagaa agtcggaaaa atttaagttc 3300
ttatacgtct tcaagtttca ttagttgtaa tcgctaaaag aaaagagagg taccagagtg 3360
aaaaggtgaa aaacagaaca ggtgattttg ggaactaaaa agtagactta tttacgatga 3420
taatcctgtg tattataatt ttctttgggg gtagataaat caataaacaa atcagtgaat 3480
attgaaattg tctaccccaa aaagacacgt tggttaaaat tcccaaaagt tatgaaattt 3540
tgtgtatgta tggttgtgaa gttgcgtgga aagtcgttga ttttattttt actgcaataa 3600
agatatacat agttctattc tttcttgttc aagttttggt agtttttttc tgtggaaaag 3660
tccacgaaaa aaataaaata tttgagtaag ggactagagc tgaagcaaga aaaaaatgga 3720
gagccaatac tcaatcaagt ttaagcaaga aaaatccaag atttagcaca aaaagaacct 3780
taacacgaca aaataggaaa tggaacagat gtttggggaa tttttgcaaa aatttccgaa 3840
aattcg 3846

Claims (7)

1. the antibiotic-free resistance recombined bacillus subtilis of one plant of expression glutamate decarboxylase, its Classification And Nomenclature is withered grass gemma Bacillus（Bacillus subtilis）SK44.001, has been preserved in China typical culture collection center, deposit number CCTCC NO：M 2016774, its be by recombinant plasmid pUB-HpaII-P43-gad-dal import host cell WB600 (dal -) and obtain Arrive, consisting of WB600/pUB-HpaII-P43-gad-dal, it is capable of synthetic amino acid array SEQ ID No.1 paddy ammonia Acid decarboxylase.
2. a kind of glutamate decarboxylase, its amino acid sequence is as shown in SEQ ID No.1.
3. the encoding gene of the glutamate decarboxylase described in claim 2, it is characterised in that：The volume of described glutamate decarboxylase Code gene source is in the Lactococcus lactis after codon optimizationLactococcus lactis subsp. lactisIL1403'sgadGene order is originalgadNucleotide sequence is as shown in SEQ ID No.2, the gad nucleotide sequences such as SEQ ID No.3 after optimization It is shown.
4. the antibiotic-free resistance recombined bacillus subtilis of glutamate decarboxylase is expressed described in claim 1, it is characterised in that： Described recombinant plasmid pUB-HpaII-P43-gad-dal, is contained containing the gad nucleic acid sequence SEQs ID after the optimization NO.3 and pUB-HpaII-P43-dal more control sequences SEQ ID No.4.
5. the construction method of the antibiotic-free resistance recombined bacillus subtilis of glutamate decarboxylase is expressed described in claim 1, It is characterized in that：
（1）Host cell WB600 (dal -) structure：WB600(dal -) be D-alanine racemase gene defect withered grass gemma Bacillus,
（a）On bacillus subtilis WB600 chromosomes D-alanine racemase gene (dal) upstream and downstream respectively choose The fragment of 900bp length is expanded two sections of homology arms by PCR as homology arm, and glue reclaim；Chooselox71-spc- lox66 resistance gene fragments as selection markers, by upstream and downstream homology arm andlox71-spc-lox66 fragments pass through fusion DNA vaccine Three sections of genes are linked together；PCR primer is transferred in bacillus subtilis WB600 competence, is containing spectinomycinSpc Flat board on screen；Due to the presence of homology arm, occur homologous recombination, knock out the D- on bacillus subtilis WB600 chromosomes Alanine racemase enzyme genedal, obtain D-alanine deficiency host WB600 (dal -)-spc；
（b）By pTSC plasmids import above-mentioned host WB600 (dal -)-spcIn, screened on the flat board containing erythromycin, in isopropyl Under base thiogalactoside IPTG induction, recombinase Cre is expressed, in the presence of recombinase Cre,lox71 Heslox66 sites Recombinate, willspcResistant gene delete, and obtain one plant of D-alanine deficiency bacillus subtilis WB600 (dal -)；
（2）Using P7, P8 as primer, using plasmid pET-22b-gad as template, PCR amplifications obtain linear fragment 1；
（3）Using P9, P10 as primer, using expression vector pUB-HpaII-P43-gad-dal as template, PCR amplifications obtain linear piece Section 2；
（4）With linear fragment 1 and linear fragment 2 primer each other, PCR amplifications obtain polymer fragment；
（5）Using GM I, GM II two-step methods prepare antibiotic-free resistance bacillus subtilis WB600 (dal -) competence is thin Born of the same parents；
（6）By step（4）Polymer fragment be transformed into step（5）In obtained competent cell, it is coated on without antibiosis Element screens without the LB flat boards of alanine and obtains recombinant bacterial strain WB600/pUB110-P43-HpaII-gad-dal.
6. recombined bacillus subtilis WB600/pUB-HpaII-P43-gad-dal fermentation process described in claim 1, it is special Levy and be：
Seed liquor culture：Single bacterium colony on picking flat board, is inoculated in 4 mL LB Tube propagation bases, 37 DEG C, 200 r/min trainings Support 12 h；The LB culture medium prescriptions are：The g/L of tryptone 10；The g/L of yeast extract 5；The g/L of sodium chloride 10；Using Deionized water is prepared, and sterilising conditions are 121 DEG C, 20 min；
Fermented and cultured：Seed liquor is transferred in 50 mL fermentation mediums by volume fraction 2%, 37 DEG C, 200 r/min cultures Final zymotic fluid is obtained after 20h；The fermentative medium formula is：The g/L of soy peptone 25；The g/L of lactose 5； Na2HPO4·12H2O 3g/L；MnSO4·H2O 0.1g/L；Prepared using deionized water, sterilising conditions are 115 DEG C, 30min.
7. the application of the recombined bacillus subtilis WB600/pUB-HpaII-P43-gad-dal described in claim 1, its feature Be express glutamate decarboxylase can be catalyzed sodium glutamate or glutamic acid production GABA GABA, can chemistry, Applied in food and pharmaceutical field.
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