CN104611396A - Glutathione production method - Google Patents
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Abstract
The invention relates to an improved glutathione production method. Recombination strain capable of efficiently conversing L-cysteine, L-glutamic acid and glycine into glutathione can be obtained by screening of recombinant expression host and selection of appropriate enzyme for over expression, and the recombination strain can effectively improve the yield of GSH(glutathione). The method can increase the yield of GSH by more than 50%.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to a kind of method of production gsh of improvement.
Background technology
Gsh (Glutathione, GSH) is a kind of medicine of important regulation of physiological functions, and application is clinically more and more wider, become medical science important there is one of medicine of mediator's body immunity function and anticancer adjuvant.The oxidation-resistance of GSH makes again its application in the food industry enjoy people to pay close attention to, in Food storage fresh-keeping and improve product special flavour, the effect improved in product nutritive value is more and more expected.Gsh is by a kind of biological activity three peptides containing γ 2 glutamyl and sulfydryl of Pidolidone, Cys, glycine condensation, in the biological phenomena that the protection etc. of protein and the synthesis of DNA, amino acid whose transhipment, cell is important, play direct or indirect effect.It is mainly distributed in animal, plant, microorganism cells, is widely used in a lot of field such as clinical medicine, sports health, food-processing.Therefore, GSH has become the focus of various countries scientist research and exploration.The method of producing gsh at present both at home and abroad mainly contains extraction process, chemical synthesis, fermentation method, enzyme process.Because fermentative Production GSH has the advantages such as bacterial classification is easy to cultivation, the convenient cheapness of raw material sources, reactions steps is simple, cost is low, transformation efficiency is high, throughput rate is fast, become the main method of current production gsh.
There is the bacterial strain of a series of production GSH in currently available technology, usually can grow under very wide culture condition and accumulate GSH, but output be general very low.Also has the problem that a lot of theory and technology is urgently captured at present, as the seed selection of superior strain and the optimization etc. of culture condition.Therefore, this area needs the novel method of research biosynthesizing further or bio-conjugate chemistry method production gsh badly.
Summary of the invention
The object of the present invention is to provide a kind of method of production gsh of improvement.
In a first aspect of the present invention, provide a kind of method of producing gsh, described method comprises:
(1) process LAN (external source) gsh bifunctional enzyme (STH) and E.C. 2.7.2.1 (ack) in Bacillus coli cells, obtain recombinant expressed cell; With
(2) cell of broken (1), by cytoclasis product and Cys, glycine, Pidolidone or its salt (as sodium salt), inorganic salt and ATP hybrid reaction, produces gsh.
In a preference, in step (1), also process LAN (external source) glutathione synthetase in Bacillus coli cells.
In another preference, in step (1), described gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene and/or glutathione synthetase-coding gene gshB are expressed in same Bacillus coli cells culture systems; Or be expressed in different intestinal bacteria culture systems.
In another preference, step (1) comprising:
A () provides a kind of recombinant Bacillus coli cells, comprise the recombinant expression cassettes of following gene in described recombinant Bacillus coli cells: gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene, and selectivity contains glutathione synthetase-coding gene gshB; Or
Recombinant Bacillus coli cells is provided, comprises: a kind of comprise the recombinant expression cassettes of gsh bifunctional enzyme encoding gene Bacillus coli cells and a kind ofly comprise the Bacillus coli cells of the recombinant expression cassettes of acetate kinase-encoding gene and/or a kind of Bacillus coli cells comprising the recombinant expression cassettes of glutathione synthetase-coding gene gshB; With
B () cultivates the recombinant Bacillus coli cells of (a), thus process LAN gsh bifunctional enzyme and E.C. 2.7.2.1.
In another preference, described intestinal bacteria are Rosetta (DE3).
In another preference, described gsh bifunctional enzyme be derive from thermophilus streptococcus (Streptococcus thermophilus) gsh bifunctional enzyme (preferably, its gene order as NCBI accession number: GU138096.1, or its according to e. coli codon preferences carry out codon optimized after sequence);
Described E.C. 2.7.2.1 derives from Lactobacillus sanfrancisco (Lactobacillus sanfranciscensis) (preferably, its gene order as NCBI accession number: AB035799.1, or its according to e. coli codon preferences carry out codon optimized after sequence); Or derive from intestinal bacteria (Escherichia coli) (preferably, its gene order as NCBI accession number: CP001509, or its according to e. coli codon preferences carry out codon optimized after sequence) E.C. 2.7.2.1.
In another preference, in step (2), described inorganic salt comprise: magnesium salts, acetyl phosphate.
In another preference, in step (2), reaction system comprises: Cys: 80 ± 40mM; Glycine: 120 ± 40mM; L-sodium: 120 ± 40mM; Seven aqueous magnesium chlorides: 40 ± 30mM; Acetylphosphate dilithium salt: 120 ± 40mM; ATP:1 ± 0.5mM; Cytoclasis product: 10 ~ 30%V/V.
In another preference, described cytoclasis product refers to: after the recombinant expressed cellular products of step (1) is centrifugal, the breakdown products after resuspended with 4 times of volume phosphoric acid buffers.
In another preference, the temperature of reaction is 30 ± 5 DEG C; Preferably 30 ± 2 DEG C.
In another preference, the pH of reaction is 7.0 ± 0.5; Preferably 7.0 ± 0.2.
In another aspect of this invention, the recombinant expression vector described in a kind of recombinant expression vector is provided to comprise the recombinant expression cassettes of following gene: gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene and/or glutathione synthetase-coding gene gshB.
In another aspect of this invention, provide a kind of Bacillus coli cells of restructuring, described Bacillus coli cells comprises described expression vector, or
Integrate the recombinant expression cassettes of following gene in the genome of described Bacillus coli cells: gsh bifunctional enzyme (STH) encoding gene and E.C. 2.7.2.1 (ack) encoding gene, and selectivity contains glutathione synthetase-coding gene gshB; Preferably, described intestinal bacteria are Rosetta (DE3).
In another aspect of this invention, providing the purposes of described recombinant expression vector or described recombinant Bacillus coli cells, is gsh for transforming Cys, Pidolidone and glycine.
In another aspect of this invention, provide a kind of test kit for the production of gsh, described test kit comprises: the Bacillus coli cells of described recombinant expression vector or described restructuring.
In a preference, the described test kit for the production of gsh, also comprises in described test kit: Cys, glycine, Pidolidone or its salt (as sodium salt), inorganic salt and ATP; Preferably, described inorganic salt comprise: seven aqueous magnesium chlorides, acetylphosphate dilithium salt.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The plasmid map of Fig. 1, recombinant plasmid pET24a-SAG.
The plasmid map of Fig. 2, recombinant plasmid pET24a-STH.
The plasmid map of Fig. 3, recombinant plasmid pET28b-gshA.
The plasmid map of Fig. 4, recombinant plasmid pET24a-gshB.
The plasmid map of Fig. 5, recombinant plasmid pTrc99a-STH.
The plasmid map of Fig. 6, recombinant plasmid pSUGAP.
The plasmid map of Fig. 7, recombinant plasmid pSUGAP-STH.
The plasmid map of Fig. 8, recombinant plasmid pET32amal.
The plasmid map of Fig. 9, recombinant plasmid pET32amal-STH.
The plasmid map of Figure 10, recombinant plasmid pET24a-ack.
The plasmid map of Figure 11, recombinant plasmid pET24a-ECack.
The plasmid map of Figure 12, recombinant plasmid pMWK.
The plasmid map of Figure 13, recombinant plasmid pET24a-gshB-ack.
The plasmid map of Figure 14, recombinant plasmid pMWK-gshB-ack.
The plasmid map of Figure 15, recombinant plasmid pTrc99a-gshB-ack-773.
The plasmid map of Figure 16, recombinant plasmid pTrc99a-ECack-773.
The protein electrophoresis of Figure 17, BL21 (DE3)/pET24a-SAG and BL21 (DE3)/pET24a-STH.Wherein, the full cell of 1-3:BL21 (DE3)/pET24a-SAG, upper cleer and peaceful precipitation; The full cell of 10-12:BL21 (DE3)/pET24a-STH, upper cleer and peaceful precipitation; The full cell of 4-6:Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack, upper cleer and peaceful precipitation; The full cell of 7-9:Rosetta (DE3)/pTrc99a-STH, upper cleer and peaceful precipitation; M is protein molecular weight standard (KDa).
The protein electrophoresis of Figure 18, BL21 (DE3)/pET24a-gshA and BL21 (DE3)/pET24a-gshB.
The precipitation of 1:BL21 (DE3)/pET28b-gshA;
The supernatant of 2:BL21 (DE3)/pET28b-gshA;
The precipitation of 3:BL21 (DE3)/pET24a-gshB;
The supernatant of 4:BL21 (DE3)/pET24a-gshB.
Embodiment
In order to improve gsh (Glutathione, GSH) output, the present inventor is through deep research, by screening recombinant expressed host and selecting suitable enzyme to carry out process LAN, obtaining Efficient Conversion Cys, Pidolidone and glycine to be the recombinant bacterial strain of gsh, applies the output that described bacterial strain can improve GSH effectively.Method of the present invention can improve gsh output more than 50%.
As used herein, as used herein, described " expression cassette " refer to include express needed for desired polypeptides (being gsh bifunctional enzyme (STH), E.C. 2.7.2.1 (ack) and/or glutathione synthetase gshB in the present invention) be necessary the gene expression system of element, usually it comprises following element: the gene order of promotor, coded polypeptide, terminator; Alternative comprises signal coding sequence etc. in addition; These elements are that operability is connected.
Therefore, the invention provides a kind of construction, described construction comprises: STH, ack and optionally comprise the expression cassette of gshB.Described expression cassette possesses all elements (comprising promotor, coding DNA and terminator etc.) needed for genetic expression, thus can intactly give expression to corresponding albumen.
Usually, described construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described construction.Described expression vector is usually also containing replication orgin and/or marker gene etc.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as apramycin (apr) resistance, Amp resistance.
Comprise the carrier of above-mentioned suitable polynucleotide sequence and suitable promotor or control sequence, may be used for transforming suitable host.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell, such as Calcium Chloride Method, electrotransformation.
It is intestinal bacteria that the present invention is used for recombinant expressed cell; Be preferably Rosetta (DE3).The present inventor finds unexpectedly, Rosetta (DE3) bacterial strain of process LAN gsh bifunctional enzyme (STH), E.C. 2.7.2.1 (ack) and/or glutathione synthetase, for the production of GSH after cracking, the output of GSH can be improved significantly.
Present invention also offers a kind of method of producing gsh, described method comprises: (1) is process LAN (external source) gsh bifunctional enzyme (STH) and E.C. 2.7.2.1 (ack) in Bacillus coli cells, obtains recombinant expressed cell; (2) cell of broken (1), by cytoclasis product and Cys, glycine, Pidolidone or its salt (as sodium salt), inorganic salt and ATP hybrid reaction, produces gsh.
In the early-stage Study of the present inventor, utilizing that escherichia coli expression is multiple may for the favourable enzyme of the production of gsh, finally finds the raising that the cellular lysate liquid of process LAN gsh bifunctional enzyme (STH) and E.C. 2.7.2.1 (ack) is conducive to output.
In addition, in early-stage Study, the present inventor has also attempted the multiple intestinal bacteria of application (as JM109, Origami (DE3), DH5 α, BL21 (DE3), Rosetta (DE3) etc.) and has carried out recombinant expressed, final finds that application Rosetta (DE3) is carried out recombinant expressedly can producing unexpected technique effect.
As optimal way of the present invention, described gsh bifunctional enzyme derives from thermophilus streptococcus (Streptococcus thermophilus); Described E.C. 2.7.2.1 derives from Lactobacillus sanfrancisco (Lactobacillus sanfranciscensis); Or derive from intestinal bacteria (Escherichia coli).Should be understood that protein variant, the bioactive fragment of the function that the albumen of reservation natural origin is substantially identical are also contained in the present invention.
The invention still further relates to the test kit for the production of gsh, described test kit comprises: for expressing the recombinant expression vector described in STH and ack and selective expression gshB; Or described test kit comprise can the Bacillus coli cells of process LAN STH and ack and selective expression gshB or product of cell lysis.Described recombinant expression vector or cell or product of cell lysis are placed in suitable container.
As optimal way of the present invention, also comprise other chemical composition of producing gsh for subsequent chemical reaction in described test kit, include but not limited to: Cys, glycine, Pidolidone or its salt (as sodium salt), inorganic salt and ATP; Preferably, described inorganic salt comprise: seven aqueous magnesium chlorides, acetylphosphate dilithium salt.
As optimal way of the present invention, in described test kit, also comprise working instructions, the concentration of various chemical reagent or expression vector or cell or product of cell lysis, usage and dosage are described.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Bacterial classification builds
Main agents: restriction enzyme and Taq archaeal dna polymerase (fermentas), T4DNA ligase enzyme, Klenow fragment, alkaline phosphatase CIAP and protein molecular weight standard (TAKARA), KOD neo plusDNA polysaccharase (TOYOBO), plasmid extraction test kit and in a small amount glue reclaim test kit (Axygen), microbiotic (the raw work in Shanghai).Primer synthesis, full genome synthesis and subclone all entrust Nanjing Genscript Biotechnology Co., Ltd. to complete.
The structure of single expression of enzymes bacterial classification of embodiment 1, product gsh (GSH)
1, the structure of pET24a-SAG
According to the gene order (NCBI accession number: AE009948.1) of the Streptococcus agalactiae source gsh bifunctional enzyme SAG reported, full genome synthesizes this sequence, two ends design restriction enzyme site NdeI and HindIII, be subcloned into the upper corresponding site of carrier pET24a (purchased from Novagen), obtain recombinant plasmid pET24a-SAG, plasmid map is shown in Fig. 1.
By recombinant plasmid pET24a-SAG Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built, obtain BL21 (DE3)/pET24a-SAG.
2, the construction and expression of pET24a-STH
According to the gene order (NCBI accession number: GU138096.1) reporting Streptococcus thermophilus strain SIIM B218 source gsh bifunctional enzyme STH, full genome synthesizes this sequence, two ends design restriction enzyme site EcoRI and XhoI, be subcloned into corresponding site on carrier pET24a, obtain recombinant plasmid pET24a-STH, plasmid map is shown in Fig. 2.
Recombinant plasmid pET24a-STH Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built and Rosetta (DE3) are obtained BL21 (DE3)/pET24a-STH and Rosetta (DE3)/pET24a-STH.
3, the structure of pET28b-gshA
To originate according to the Escherichia coli BL21 (DE3) reported the gene order (NCBI accession number: AM946981.2) of Glutamate-cysteine ligase gene gshA, design primer is as follows: gshA-NcoI-F:CATGCCATGGGAATCCCGGACGTATCACAGGC (SEQ ID NO:1), gshA-BamHI-R:CGGGATCCTCAGGCGTGTTTTTCCAGCC (SEQ ID NO:2) is template amplification gshA fragment with BL21 (DE3) genomic dna.PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1X KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH
2o to total system 50 μ L.Pcr amplification condition is: 94 DEG C of 2min, 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 45s, repeats 30 circulations, 68 DEG C of 10min.After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of about an about 1.5kb detected, reclaim test kit with glue to reclaim, NcoI and BamHI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: PCR primer 42 μ L, 10 × Tango buffer5 μ L, NcoI1.5 μ L, BamHI1.5 μ L.Glue reclaims test kit and crosses column purification recovery.Reclaim expression vector pET28b (purchased from Novagen) that product and same enzyme cut process to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, Transformed E .coli DH5 α competent cell, obtain recombinant plasmid pET28b-gshA, plasmid map is shown in Fig. 3, transforms BL21 (DE3) and obtain BL21 (DE3)/pET28b-gshA after extraction.
4, the structure of pET24a-gshB
To originate according to the Escherichia coli BL21 (DE3) reported the gene order (NCBI accession number: AM946981.2) of Glutatione synthetase gene gshB, design primer is as follows: gshB-NdeI-F:GGAATTCCATATGatcaagctcggcatcgt (SEQ ID NO:3), gshB-BamHI-R:CGGGATCCttactgctgctgtaaacgtg (SEQ ID NO:4) is template amplification gshB fragment with BL21 (DE3) genomic dna.PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1 × KODneo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH
2o to total system 50 μ L.Pcr amplification condition is: 94 DEG C of 2min, 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 30s, repeats 30 circulations, 68 DEG C of 10min.After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of about an about 1kb detected, reclaim test kit with glue to reclaim, NdeI and BamHI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: PCR primer 37 μ L, 10 × Tango buffer10 μ L, NdeI1.5 μ L, BamHI1.5 μ L.Glue reclaims test kit and crosses column purification recovery.Reclaim expression vector pET24a that product and same enzyme cut process to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, Transformed E .coli DH5 α competent cell, obtain recombinant plasmid pET24a-gshB, plasmid map is shown in Fig. 4, transforms BL21 (DE3) and obtain BL21 (DE3)/pET24a-gshB after extraction.
5, the protein electrophoresis of above-mentioned bacterial classification
The bacterial classification built is inoculated into respectively in the LB liquid nutrient medium containing 100 μ g/mL kantlex, after 37 DEG C of 220rpm overnight incubation, (containing 100 μ g/mL kantlex) in fresh TB substratum are inoculated into by 1% (v/v) inoculum size, 37 DEG C of 220rpm are cultured to OD600=5-6, adding final concentration is 0.2mMIPTG, 28 DEG C of overnight induction.
Getting after the bacterium liquid 10ml after induction collects thalline uses 20ml0.1M PBS damping fluid (pH7.0) resuspended, ultrasonication, arrange respectively full enchylema (1ml breaks cytosol), supernatant liquor (1ml break cytosol centrifugal after get supernatant), precipitated liquid (1ml break cytosol centrifugal after abandon supernatant, add the resuspended precipitation of 1ml damping fluid) three increment product, each for electrophoresis Sample 20 μ l are mixed with 5 μ l5 × sample buffers and boils 5 minutes, get supernatant 10 μ L after centrifugal and carry out SDS-PAGE analysis.Adopt 5% concentrated glue and 12% separation gel, electrophoresis terminates rear gel coomassie brilliant blue R250 and dyes.Result is as Figure 17-18.
6, the construction and expression of pTrc99a-STH
Recombinant plasmid pET24a-STH EcoRI and XhoI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: plasmid 37 μ L, 10 × Tango buffer10 μ L, EcoRI1.5 μ L, XhoI1.5 μ L.Gel electrophoresis glue reclaims test kit and reclaims 2.2kb STH fragment.
Carrier pTrc99a (purchased from Pharmacia) is with EcoRI and SalI in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: plasmid 15 μ L, 10 × Buffer O5 μ L, EcoRI1.5 μ L, SalI1.5 μ L, and moisturizing is to total system 50 μ L.Glue recovery test kit is crossed post and is returned pTrc99a endonuclease bamhi.
Utilize XhoI with SalI isocaudarner to be connected, above-mentioned two fragments to be connected in 16 DEG C with T4DNA ligase enzyme and spend the night, connect product conversion E.coli DH5 α competent cell, coating Amp is dull and stereotyped.
Choose single bacterium colony from transformation plate and connect LB Tube propagation, with plasmid extraction test kit extracting plasmid, obtain recombinant plasmid pTrc99a-STH, plasmid map is shown in Fig. 5.Afterwards with the checking of EcoRI and HindIII double digestion, if 2.2kb fragment can be cut out, be correct.
By the recombinant plasmid pTrc99a-STH Calcium Chloride Method transformation of E. coli expressive host JM109 (purchased from the raw work in Shanghai) respectively built, Origami (DE3) (purchased from Novagen) and Rosetta (DE3) (purchased from Novagen) obtains JM109/pTrc99a-STH, Origami (DE3)/pTrc99a-STH and Rosetta (DE3)/pTrc99a-STH.
7, the construction and expression of pSUGAP-STH
Plasmid pSU2718 (Martinez, E. etc., Gene1988,68,159) use SacI and Sma I in 37 DEG C of double digestion plasmid 3-6h, the enzyme system of cutting is: plasmid 37 μ L, 10 × Tango buffer10 μ L, SacI1.5 μ L, SmaI1.5 μ L, glue reclaims the pSU2718 fragment that test kit reclaims 2.3kb.
Design primer is as follows: ecgapup:GGTACCGAGCTCGAGGCGAGTCAGTCGCGTAATGC (SEQ ID NO:5); Ecgapdn:TTTCCCGGGTTAATTAAGATCTATATTCCACCAGCTATTTGTTAG (SEQ ID NO:6), from genome of E.coli, the 0.2kb fragment comprising GAP promotor is isolated by PCR, PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neoplus1U, mends ddH
2o to total system 50 μ L.Temperature condition is: 94 DEG C of 2min; 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 20s, repeat 30 circulations; 68 DEG C of 10min.After PCR reaction terminates, agarose gel electrophoresis is identified, glue reclaims test kit and reclaims GAP fragment, with SacI and SmaI in 37 DEG C of double digestion 3-6h, the enzyme system of cutting is: GAP fragment 37 μ L, 10 × Tango buffer10 μ L, SacI1.5 μ L, SmaI1.5 μ L, the pSU2718 fragment of the 2.3kb obtained with above-mentioned recovery after glue recovery test kit crosses post recovery is connected in 16 DEG C of water-baths under the effect of T4 ligase enzyme spends the night, Transformed E .coli DH5 α competent cell, coating Cm is dull and stereotyped, 37 DEG C of overnight incubation.Select transformant to connect LB Tube propagation and spend the night, with the checking of test kit extracting plasmid, obtain pSUGAP plasmid, plasmid map is shown in Fig. 6.
PET24a-STH NheI and XhoI does substep enzyme in 37 DEG C and cuts, and glue reclaims about 2.3kb STH fragment, and pSUGAP XbaI and SalI (2 × Buffer Tango), in 37 DEG C of double digestion 3-6h, crosses column purification and reclaim pSUGAP carrier segments.These two fragments to connect in 16 DEG C with T4DNA ligase enzyme spends the night, and transform DH5 α competent cell, coating Cm is dull and stereotyped, 37 DEG C of overnight incubation.Choose single bacterium colony from transformation plate and connect LB Tube propagation, the BamHI single endonuclease digestion checking of upgrading grain, correct recombinant clone can cut out about 1kb fragment, and plasmid map is shown in Fig. 7.
Recombinant plasmid pSUGAP-STH Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built is obtained BL21 (DE3)/pSUGAP-STH.
8, the construction and expression of pET32amal-STH
PMal-c2x (purchased from New England Biolabs) is with NdeI and HindIII in 37 DEG C of double digestion 3-6h, and the enzyme system of cutting is: plasmid 42 μ L, 10 × Buffer R5 μ L, NdeI1.5 μ L, HindIII1.5 μ L.Agarose gel electrophoresis detects blend compounds and reclaims test kit recovery 1.2kb mal fragment, to connect in 16 DEG C with T4DNA ligase enzyme with pET32a (purchased from the novagen) fragment of same double digestion process and spend the night, transform DH5 α competent cell, coating Amp is dull and stereotyped, 37 DEG C of overnight incubation.Choose single bacterium colony from transformation plate and connect LB Tube propagation, the NdeI/HindIII double digestion checking of upgrading grain, correct recombinant clone can cut out about 1.2kb fragment, and obtain pET32amal plasmid, plasmid map is shown in Fig. 8.
PET24a-STH EcoRI and XhoI double digestion in 37 DEG C of water-baths spends the night, and the enzyme system of cutting is: plasmid 37 μ L, 10 × Buffer Tango10 μ L, EcoRI1.5 μ L, XhoI1.5 μ L.Glue reclaims about 2.2kb STH fragment, and to connect in 16 DEG C with T4DNA ligase enzyme with the pET32amal fragment of same double digestion process and spend the night, transform DH5 α competent cell, coating Amp is dull and stereotyped, 37 DEG C of overnight incubation.Choose single bacterium colony from transformation plate and connect LB Tube propagation, the EcoRI/XhoI double digestion checking of upgrading grain, correct recombinant clone can cut out about 2.2kb large fragment.Plasmid map is shown in Fig. 9.
By recombinant plasmid pET32amal-STH Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) respectively built, Origami (DE3) and Rosetta (DE3), obtain BL21 (DE3)/pET32amal-STH, Origami (DE3)/pET32amal-STH and Rosetta (DE3)/pET32amal-STH.
9, the construction and expression of pET24a-ack
According to the gene order (NCBI accession number: AB035799.1) of the Lactobacillus sanfranciscensis source Acetokinase gene ack reported, full genome synthesizes this sequence, two ends design restriction enzyme site NdeI and BamHI, be subcloned into corresponding site on carrier pET24a, obtain recombinant plasmid pET24a-ack, plasmid map is shown in Figure 10.
Recombinant plasmid Calcium Chloride Method transformation of E. coli expressive host BL21 (DE3) built is expressed, obtains E.C. 2.7.2.1 and express bacterial classification BL21 (DE3)/pET24a-ack.
10, the construction and expression of pET24a-ECack
To originate according to the Escherichia coli BL21 (DE3) reported the gene order (NCBI accession number: CP001509.3) of Acetokinase gene ECack, design primer is as follows: ECack-NdeI-F:GGAATTCCATATGTCGAGTAAGTTAGTAC (SEQ ID NO:7), ECack-BamHI-R:CGGGATCCTCAGGCAGTCAGGCGGCT (SEQ ID NO:8) is template amplification ECack fragment with BL21 (DE3) genomic dna.PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH
2o to total system 50 μ L.Pcr amplification condition is: 94 DEG C of 2min, 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 45s, repeats 30 circulations, 68 DEG C of 10min.After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of about an about 1.2kb detected, reclaim test kit with glue to reclaim, NdeI and BamHI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: PCR primer 37 μ L, 10 × Buffer Tango10 μ L, NdeI1.5 μ L, BamHI1.5 μ L.Glue reclaims test kit and crosses column purification recovery.
Reclaim expression vector pET24a that product and same enzyme cut process to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, Transformed E .coli DH5 α competent cell, obtain recombinant plasmid pET24a-ECack, plasmid map is shown in Figure 11, transforms BL21 (DE3) and obtain BL21 (DE3)/pET24a-ECack after extraction.
11, the codon optimized expression of STH
To the gene order (Streptococcus thermophilus strain SIIM B218 originates) of STH, according to colibacillary codon-bias carry out codon optimized after, again synthesis obtains STH-opt, is then integrated into pET24a Plastid transformation BL21 (DE3) and obtains BL21 (DE3)/pET24a-STH-opt.
STH-opt sequence following (SEQ ID NO:9):
atgaccctgaaccaactgctgcaaaaactggaagcgacctcgccgatcctgcaagccaacttcggtattgaacgtgaaagcctgcgtgtcgatcgccagggtcaactggtgcataccccgcacccgagttgcctgggtgcccgttccttccatccgtatatccagaccgatttctgtgaatttcaaatggaactgatcacgccggttgcgaaaagcaccacggaagcccgtcgctttctgggcgccatcaccgatgtcgcaggtcgctcaattgctacggacgaagtgctgtggccgctgagcatgccgccgcgtctgaaagcagaagaaattcaggtggctcaactggaaaacgatttcgaacgtcattatcgcaattacctggcagaaaaatatggcaccaagctgcaggctattagcggtatccactacaacatggaactgggcaaagatctggttgaagcactgttccaggaatctggtcaaaccgacatgatcgcttttaaaaacgcgctgtatctgaagctggcgcagaattatctgcgttaccgctgggtgatcacgtacctgtttggtgccagtccgattgcagaacagggctttttcgatcaagaagtgccggaaccggttcgtagcttccgcaactctgaccatggttatgtgaacaaagaagaaatccaggtgtctttcgtttccctggaagattatgtttccgcgattgaaacctacatcgaacagggcgacctgaacgcagaaaaggaattttacagtgctgttcgtttccgcggtcaaaaagtcaatcgctccttcctggataagggcattacctacctggaatttcgcaacttcgacctgaatccgtttgaacgtattggtatctcacagaccacgatggatacggtccacctgctgatcctggcctttctgtggctggattcgccggaaaacgtggaccaggctctggcgcaaggccatgcgctgaatgaaaaaattgcgctgagtcacccgctggaaccgctgccgtccgaagccaagacccaggatatcgtgacggccctggaccagctggttcaacatttcggcctgggtgattatcaccaggacctggtcaaacaagtgaaggcggcctttgcagatccgaaccagaccctgagcgctcaactgctgccgtacattaaggataagtctctggctgaatttgcgctgaataaagccctggcatatcatgattacgactggaccgcccactatgcactgaaaggttacgaagaaatggaactgagcacgcagatgctgctgtttgatgcgattcaaaaaggcatccatttcgaaattctggatgaacaggaccaatttctgaagctgtggcatcaggatcacgttgaatatgtcaaaaacggtaatatgacctctaaggacaactacgtggttccgctggctatggcgaataaaaccgttacgaaaaagattctggccgatgcaggctttccggtcccgtcaggtgacgaatttacctcgctggaagaaggcctggcctattacccgctgatcaaagataagcagattgtcgtgaaaccgaagtcaacgaactttggcctgggtattagcatcttccaggaaccggcgtctctggataattatcaaaaagccctggaaattgctttcgcggaagacacctcggttctggtcgaagaatttatcccgggtacggaataccgtttctttattctggatggccgctgcgaagcggtcctgctgcgtgtggcagctaatgttatcggcgatggtaaacataccattcgcgaactggtggctcagaagaacgcgaatccgctgcgtggtcgtgatcaccgtagcccgctggaaattatcgaactgggcgacatcgaacagctgatgctggcacagcaaggttataccccggatgacattctgccggaaggcaaaaaggttaacctgcgtcgcaactcaaatatctcgacgggcggtgatagtattgacatcaccgaaacgatggatagctcttatcaggaactggccgcggcaatggccaccagcatgggtgcatgggcatgtggtgtggatctgattatcccggacgaaacccagatcgccacgaaagaaaatccgcattgcacctgtattgaactgaactttaatccgagcatgtatatgcacacgtactgtgcggaaggcccgggtcaggcgatcacgacgaagattctggataaactgttcccggaaattgtggcaggtcaaacgtaa
12, the GSH determination of yield that shares of the different strain of aforementioned structure and BL21 (DE3)/pET24a-ack
The fermentation process of various bacterial classification: LB substratum (peptone 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, pH7.2); TB substratum (peptone 12g/L, yeast extract 24g/L, glycerine 5g/L, KH
2pO
42.13g/L, K
2hPO
43H
2o16.43g/L, pH7.0-7.5); 121 DEG C of autoclave sterilizations 20 minutes.Shake flask culture conditions: the bacterial classification built is inoculated into respectively containing in 100 μ g/mL separately corresponding antibiotic LB liquid nutrient medium, after 37 DEG C of 220rpm overnight incubation, (containing 100 μ g/mL corresponding microbiotic separately) in fresh TB substratum is inoculated into by 1% inoculum size, 37 DEG C of 220rpm are cultured to OD600=5-6, adding final concentration is 0.2mM IPTG, 28 DEG C of overnight induction.
Bacterial cell disruption liquid processing method: the phosphoric acid buffer (0.1mM, pH7.2) of the centrifugal acquisition thalline 4 times of volumes that ferment is resuspended, then ultrasonication.
Produce GSH reaction system: reaction final volume 5ml (phosphoric acid buffer 0.1mM, pH6.8): Cys: 80mM, glycine: 120mM, L-sodium: 120mM, seven aqueous magnesium chlorides: 40mM, acetylphosphate dilithium salt: 120mM, ATP:1mM, E.C. 2.7.2.1 thalline (BL21 (DE3)/pET24a-ack) broken liquid 0.4% (V/V), (wherein BL21 (DE3)/pET28b-gshA and BL21 (the DE3)/pET24a-gshB's broken liquid of each recombinant bacterium of aforementioned structure: 10%V/V lives according to different ratios mixed determining enzyme, both total consumptions are 10% (V/V)).First successively chemical reagent is added phosphoric acid buffer by above-mentioned formula, then adjust pH6.8, add bacterial cell disruption liquid, load test tube, seal after logical nitrogen, 30 DEG C, 150rpm reacts, react after 30 minutes, get 500 μ l samples and add in EP pipe, then add 500 μ l10% hydrochloric acid stopped reactions.
The yield data recorded is as table 1.
Table 1
| Bacterial classification | 30min GSH content |
| Rosetta(DE3)/pET24a-STH | 0.8g/L |
| JM109/pTrc99a-STH | 3.6g/L |
| Origami(DE3)/pTrc99a-STH | 5.3g/L |
| Rosetta(DE3)/pTrc99a-STH | 7.5g/L |
| DH5α/pSUGAP-STH | 0g/L |
| BL21(DE3)/pSUGAP-STH | 0g/L |
| BL21(DE3)/pET32amal-STH | 0.8g/L |
| Origami(DE3)/pET32amal-STH | 0g/L |
| Rosetta(DE3)/pET32amal-STH | 0.4g/L |
| BL21(DE3)/pET24a-STH | 2.7g/L |
| BL21(DE3)/pET24a-SAG | 6.8g/L |
| BL21(DE3)/pET24a-STH-opt | 5.9g/L |
| gshA/gshB(1：1) | 0.8g/L |
| gshA/gshB(2：1) | 1.8g/L |
| gshA/gshB(4：1) | 2.0g/L |
| gshA/gshB(8：1) | 0.8g/L |
From above-mentioned data, the output that Rosetta (DE3)/pTrc99a-STH and BL21 (DE3)/pET24a-ack shares is the highest.
13, the comparison of two kinds of ACK
By the conversion of Rosetta (DE3)/pTrc99a-STH, compare different sources ack to the situation that affects transformed (BL21 (DE3)/pET24a-ack and BL21 (DE3)/pET24a-ECack (large intestine source ack).
Fermentation and bacterial cell disruption technique are with embodiment 1-12
Comparison condition: reaction final volume 50ml (phosphoric acid buffer 0.1mM, pH6.8): Cys: 80mM, glycine: 120mM, L-sodium: 120mM, seven aqueous magnesium chlorides: 40mM, acetylphosphate dilithium salt: 120mM, ATP:1mM, different E.C. 2.7.2.1 bacterial cell disruption liquid 0.4%V/V, Rosetta (DE3)/pTrc99a-STH bacterial cell disruption liquid: 10%V/V.First successively chemical reagent is added phosphoric acid buffer by above-mentioned formula, then adjust pH6.8, add bacterial cell disruption liquid, load 250ml shaking flask, seal after logical nitrogen, 30 DEG C, 150rpm reacts.React after 30 minutes, get 500 μ l samples and add in EP pipe, then add 500 μ l10% hydrochloric acid stopped reactions.
Result is as table 2.
Table 2
| 0.5h GSH output | |
| BL21(DE3)/pET24a-ack | 8.26g/L |
| BL21(DE3)/pET24a-ECack | 8.92g/L |
By contrast, the present inventor finds that two kinds of kinases can well transform when biomass uses little.
14, Rosetta (DE3)/pTrc99a-STH, BL21 (DE3)/pET24a-SAG and BL21 (DE3)/pET28b-gshA and BL21 (DE3)/pET24a-gshB and BL21 (DE3)/pET24a-ack mixing transforms the comparison of producing GSH
Zymotechnique and bacterial cell disruption technique are with embodiment 1-12.
Conversion process: reaction final volume 50ml (phosphoric acid buffer 0.1mM, pH7.2): Cys: 80mM, glycine: 120mM, L-sodium: 120mM, seven aqueous magnesium chlorides: 40mM, acetylphosphate dilithium salt: 120mM, ATP:1mM, Rosetta (DE3)/pTrc99a-STH, (wherein BL21 (DE3)/pET28b-gshA and BL21 (the DE3)/pET24a-gshB's bacterial cell disruption liquid of BL21 (DE3)/pET24a-SAG and BL21 (DE3)/pET28b-gshA and BL21 (DE3)/pET24a-gshB: 20%V/V lives according to different ratios mixed determining enzyme, both total consumptions are 20%V/V), E.C. 2.7.2.1 thalline (BL21 (DE3)/pET24a-ack) broken liquid 0.4%V/V.First successively chemical reagent is added phosphoric acid buffer by above-mentioned formula, then adjust pH7.2, add bacterial cell disruption liquid, load 250ml shaking flask, seal after logical nitrogen, 30 DEG C, 150rpm reacts, and gets 500 μ l samples and adds in EP pipe, then add 500 μ l10% hydrochloric acid stopped reactions.
Result is as table 3.
Table 3
| Bacterial classification | 1h GSH output |
| Rosetta(DE3)/pTrc99a-STH | 16.9g/L |
| BL21(DE3)/pET24a-SAG | 7.7g/L |
| gshA/gshB(1：1) | 7.5g/L |
| gshA/gshB(2：1) | 8.5g/L |
| gshA/gshB(4：1) | 7.5g/L |
From above-mentioned data, the output that Rosetta (DE3)/pTrc99a-STH and BL21 (DE3)/pET24a-ack shares is the highest.
The structure of the combination expression of enzymes bacterial classification of embodiment 2, product GSH
1, the structure of STH and gshB-ack coexpression bacterial classification
(1) structure of pMWK-gshB-ack
With plasmid pPIC3.5K (purchased from Invitrogen) for masterplate, design primer is as follows: Kandn:CCAACCAATTAACCAATTCTGATTAG (SEQ ID NO:10), Kanup:CCTGCAGGGGGGGGGGGAAAGCCACGTTGTGTC (SEQ ID NO:11), pcr amplification is about 0.9kb Kan fragment.PCR reaction system comprises: each 0.3 μM of primer, template 50ng, 1 × KODneo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH2O to total system 50 μ L.Temperature condition is: 94 DEG C of 2min; 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 30s, repeat 30 circulations; 68 DEG C of 10min.After PCR reaction terminates, agarose gel electrophoresis is identified, glue reclaims test kit and reclaims kan fragment.Plasmid pMW118 (purchased from Nippon Gene, Toyama, Japan) is with ApaLI and EcoO1109I in 37 DEG C of double digestion 3-6h, and the enzyme system of cutting is: plasmid 37 μ L, 10 × BufferTango10 μ L, ApaLI1.5 μ L, EcoO1109I1.5 μ L.Agarose gel electrophoresis detects blend compounds and reclaims test kit recovery 2.7kb fragment, fills 3 ' recessed end by Klenow fragment.Kan fragment and pMW118 are filled fragment to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, transform DH5 α competent cell, coating Kan is dull and stereotyped, 37 DEG C of overnight incubation.Choosing single bacterium colony from transformation plate utilizes KanF/M13F-47 to be bacterium colony PCR checking (kanF:TCGGAATCGCAGACCGATAC (SEQ ID NO:12), M13F-47:CGCCAGGGTTTTCCCAGTCACGAC (SEQ ID NO:13)), if 500bp fragment can be amplified, be be connected in the right direction, obtain pMWK, plasmid map is shown in Figure 12.
Recombinant plasmid pET24a-ack BglII and BamHI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: plasmid 37 μ L, 10 × Tango buffer10 μ L, BglII1.5 μ L, BamHI1.5 μ L.Gel electrophoresis glue reclaims test kit and reclaims 1.2kb ack fragment.
Recombinant plasmid pET24a-gshB BamHI is in 37 DEG C of single endonuclease digestion 3-6 hour, and the enzyme system of cutting comprises: plasmid 30 μ L, 10 × BamHI buffer5 μ L, BamHI1.5 μ L, and moisturizing is to total system 50 μ L.Glue reclaims test kit and reclaims 6.2kb carrier segments.Reclaim product alkaline phosphatase CIAP dephosphorylation in 37 DEG C of water-baths and react 1 hour, reaction system is: 10 × Alkaline Phospharase buffer5 μ L, pET24a-gshB single endonuclease digestion sheet 44 μ L, CIAP1 μ L.Glue reclaims test kit and crosses column purification dephosphorylation fragment.
Above-mentioned two fragments to be connected in 16 DEG C with T4DNA ligase enzyme and spend the night, connect product conversion E.coliDH5 α competent cell, coating Kan is dull and stereotyped.
Choose single bacterium colony from transformation plate and connect LB Tube propagation, with plasmid extraction test kit extracting plasmid, obtain recombinant plasmid pET24a-gshB-ack (Figure 13).Afterwards with the checking of BglII and BamHI double digestion, if 2.3kb fragment can be cut out, be correct.
With recombinant plasmid pET24a-gshB-ack for template design primer amplification gshB-ack fragment.Design of primers is as follows: gshB-SalI-F:ACGCGTCGACATGATCAAGCTCGGCATC (SEQ ID NO:14), ack-BamHI-R:CGGGATCCTTATTTATTTGCTAATGT (SEQ ID NO:15).PCR reaction system comprises: each 0.3 μM of primer, template 50ng, 1 × KOD neo plus buffer, 0.2mMdNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH2O to total system 50 μ L.Pcr amplification condition is: 94 DEG C of 2min, 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 1min15s, repeats 30 circulations, 68 DEG C of 10min.After PCR reaction terminates, analyze with agarose gel electrophoresis, the specific band of about an about 2.3kb detected, reclaim test kit with glue to reclaim, SalI and BamHI is in 37 DEG C of double digestion 3-6 hour, and the enzyme system of cutting comprises: PCR primer 42 μ L, 10 × Buffer BamHI5 μ L, SalI1.5 μ L, BamHI1.5 μ L.Glue reclaims test kit and crosses column purification recovery.
Plasmid pMWK uses SalI and BamHI in 37 DEG C of double digestion 3-6 hour equally, and glue reclaims test kit and reclaims 3.6kb carrier segments.
The gshB-ack fragment of recovery and pMWK fragment to be connected in 16 DEG C with T4DNA ligase enzyme and spend the night, connect product conversion E.coli DH5 α competent cell, coating Kan is dull and stereotyped.
Choose single bacterium colony from transformation plate and connect LB Tube propagation, with plasmid extraction test kit extracting plasmid, use SalI/BamHI double digestion, if it is correct for cutting out 2.3kb fragment, obtain recombinant plasmid pMWK-gshB-ack, plasmid map is shown in Figure 14.
(2) structure of coexpression bacterial classification
By plasmid pMWK-gshB-ack Calcium Chloride Method transformation of E. coli host Rosetta (DE3)/pTrc99a-STH (the embodiment 1-6 acquisition) competent cell of above-mentioned structure, the LB of coating containing Cm/Amp/Kan is dull and stereotyped, the exactness that the checking of picking mono-clonal extracting plasmid transforms, obtains coexpression bacterial classification Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack.
2, the expression of STH in the escherichia coli host Rosetta (DE3) integrating gshB-ack
The transformation of escherichia coli expression host: gshB-ack is incorporated into the genomic yaiT site of Rosetta (DE3):
(1) structure of pTrc99a-gshB-ack-773
Aforementioned recombinant plasmid pET24a-gshB NdeI/BamHI (2 × Buffer Tango) double digestion built, glue reclaims about 1kb gshB fragment, is cloned into corresponding site on carrier pET28b, obtains recombinant plasmid pET28b-gshB.
PET28b-gshB NcoI/BamHI (1 × Buffer Tango) double digestion, glue reclaims about 1kb gshB fragment, is cloned into corresponding site on carrier pTrc99a, obtains recombinant plasmid pTrc99a-gshB.
Aforementioned recombinant plasmid pET24a-ack XbaI/SalI (2 × Buffer Tango) double digestion built, glue reclaims 1.2kb ack fragment, is cloned into corresponding site on carrier pTrc99a-gshB, obtains recombinant plasmid pTrc99a-gshB-ack.
Plasmid pIJ773 is (see B Gust, GL Challis, K Fowler, T Kieser, KF Chater (2003) Proc Natl Acad Sci U S A.100 (4): 1541-6.) use EcoRI/SalI (Buffer O) double digestion, glue reclaims the apramycin resistance gene (1.5kb) that FRT site is contained on both sides, this is reclaimed pTc99a-gshB-ack that fragment and same enzyme cut process to connect in 16 DEG C with T4DNA ligase enzyme and spend the night, connect product conversion E.coliDH5 α competent cell, coating Amp/Apr is dull and stereotyped.The exactness that the checking of picking mono-clonal extracting plasmid transforms, obtain recombinant plasmid pTrc99a-gshB-ack-773, plasmid map is shown in Figure 15.
(2) be template amplification integration site yaiT gene upstream and downstream homologous sequence with Rosetta (DE3) genome
yaiT-SwaI-F：atttaaattgaaccattcgtctgtaacgcagc(SEQ ID NO:16)
yaiT-gshB-R：tcgatgataagctgtcaaacatcaagctgattagcagtac(SEQ ID NO:17)
773-yaiT-F：ctttttgcgtttctacaaacatccatagcagcgattacgt(SEQ ID NO:18)
yaiT-SwaI-R：atttaaatgtcagacgagtaccaacggtgtct(SEQ ID NO:19)
With Rosetta (DE3) genomic dna for template, two couples of primer yaiT-SwaI-F and yaiT-gshB-R, 773-yaiT-F and yaiT-SwaI-R increase 400bp upstream homology arm yaiT-up and 400bp downstream homology arm yaiT-dn respectively.
PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH2O to total system 50 μ L.PCR reaction conditions is: 94 DEG C of 2min; 98 DEG C of 10s, 65 DEG C of 30s, 68 DEG C of 30s, after this each is taken turns circulation annealing temperature and reduces by 1 DEG C, 20 circulations, last 45 DEG C 10 circulations; 68 DEG C of 10min.Running gel reclaims 400bp yaiT-up fragment and 400bp yaiT-dn fragment.
(3) take pTrc99a-gshB-ack-773 as template, yaiT-gshB-F and yaiT-773-R is primer, comprises the long segment ptrc-gshb-ack-773 of promotor, integrator gene and the resistant gene with FRT site with KOD enzymatic amplification.
yaiT-gshB-F：gtactgctaatcagcttgatgtttgacagcttatcatcga(SEQ ID NO:20)
773-yaiT-R：acgtaatcgctgctatggatgtttgtagaaacgcaaaaag(SEQ ID NO:21)
PCR reaction system comprises: each 0.3 μM of primer, template 50ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH2O to total system 50 μ L.PCR reaction conditions is: 94 DEG C, 2min; 98 DEG C of 10s, 68 DEG C of 2min, repeat 30 circulations; 68 DEG C of 10min.Running gel reclaims 4kb object fragment.
(4) OVERLAP-PCR splices three fragments, obtains integrating box
Electrophoresis detection yaiT-up, yaiT-dn, ptrc-gshB-ack-773 tri-fragment concentrations, as far as possible guarantee three fragment equimolar ratio.
PCR reaction system comprises: 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, each 0.3 μM of each 0.3 μM of 2%DMSO, yaiT-up, yaiT-dn and ptrc-gshB-ack, yabp-SwaI-F and yabp-SwaI-R, KOD neo plus1U, moisturizing, to being totally 50 μ L, 6 is managed totally.
PCR reaction conditions is: 94 DEG C of 2min; 98 DEG C of 10s, 65 DEG C of 30s, 68 DEG C of 2min30s, after this each is taken turns circulation annealing temperature and reduces by 1 DEG C, 20 circulations, last 45 DEG C 10 circulations; 68 DEG C of 10min.
Running gel reclaims about 5kb large fragment, for ptrc-gshB-ack-773 integrates box, is concentrated into 10 μ about L.
(5) Red homologous recombination integrates Rosetta (DE3)
To Rosetta (DE3) strain inoculation having pKD46 be transformed, 30 DEG C of overnight incubation.Next day is inoculated in 50mL SOB substratum (containing 100 μ g/mL Amp) with 1:50, and add 20mM L-arabinose, 30 DEG C are cultured to OD600=0.6 simultaneously, and Exo, Bet and Gam tri-albumen on pKD46 are given full expression to.Precooling 10min on ice, bacterium liquid in 4 DEG C, centrifugal 10min abandons supernatant to 4000rpm, with the 10% glycerine centrifuge washing 3 times of precooling, is condensed into the competent cell of 100-200 μ L.
Get 2-3uL integration box fragment and add competent cell, mixing, proceed to 0.2cm and shock by electricity in cup, do electricity and transform, voltage 2.5kV with Bio-rad electric shock instrument, shock by electricity time 4-5s.Add rapidly 1mL LB after electric shock, 37 DEG C, 200rpm recovery 1h, coating Apr+Cm is dull and stereotyped, 37 DEG C of overnight incubation.
Be bacterium colony PCR with 4 pairs of primers respectively and identify the apramycin resistance clone grown:
yaiTupup100：AGGGCGTTGGATTAAGTCTGTC(SEQ ID NO:22)
gshBdn：atcacgatgccgagcttgat(SEQ ID NO:23)
Correct integron can expand 1.2kb fragment;
pTrcHisrev-F：agcctgatacagattaaatc(SEQ ID NO:24)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:25)
Correct integron can expand 1.2kb fragment;
Apr-F：cgccattcgtattgcacg(SEQ ID NO:26)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:27)
Correct integron can expand 1.7kb fragment;
Contrast Rosetta (DE3) all can not expand band.
In the integron of screening, can expand bright band with pair of primers, size is correct; Can expand very light band with second and third to primer, but size is correct; Contrast does not all expand band.Obtain intestinal bacteria transformation host Rosetta (DE3):: gshB-ack.
(6) STH is at Rosetta (DE3):: the expression in gshB-ack
The recombinant plasmid pTrc99a-STH Calcium Chloride Method of aforementioned structure is transformed above-mentioned intestinal bacteria transformation host Rosetta (DE3):: gshB-ack, obtains Rosetta (DE3):: gshB-ack/pTrc99a-STH.
3, the expression of STH in the escherichia coli host Rosetta (DE3) integrating ECack
The Acetokinase gene ECack (NCBI accession number: CP001509.3) of Escherichia coli is incorporated into the genomic yaiT site of Rosetta (DE3).
(1) structure of pTrc99a-ECack-773
With genome of E.coli DNA for template, design primer amplification 1.2kb ECack fragment.
ECack-NcoI-F：CATGCCATGGgatcgagtaagttagtactg(SEQ ID NO:28)
ECack-BamHI-R：CGGGATCCtcaggcagtcaggcggct(SEQ ID NO:29)
PCR reaction system comprises: each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO
4, KOD neo plus1U, mends ddH2O to total system 50 μ L.PCR reaction conditions is: 94 DEG C, 2min; 98 DEG C of 10s, 68 DEG C of 45s, repeat 30 circulations; 68 DEG C of 10min.
Running gel reclaims 1.2kb object fragment, reclaim with purifying after NcoI/BamHI (1 × Buffer Tango) double digestion process, the pTrc99a-gshB-ack-773 fragment of cutting process with same enzyme to connect in 16 DEG C with T4DNA ligase enzyme spends the night, Calcium Chloride Method transforms DH5 α, coating Amp plate incubated overnight.Choose mono-clonal NcoI/BamHI to verify, if it is correct for cutting out 1.2kb fragment, obtain recombinant plasmid pTrc99a-ECack-773, plasmid map is as Figure 16.
(2) preparation of box fragment is integrated with apramycin resistance and Red homologous recombination integrates the method for Rosetta (DE3) with abovementioned steps 2 (2)-(5).
Be bacterium colony PCR with 2 pairs of primers respectively and identify the apramycin resistance clone grown:
yaiTupup100：AGGGCGTTGGATTAAGTCTGTC(SEQ ID NO:30)
ECackdn：agtactaacttactcgatcc(SEQ ID NO:31)
Correct integron can expand 1.2kb fragment;
Apr-F：cgccattcgtattgcacg(SEQ ID NO:32)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:33)
Correct integron can expand 1.7kb fragment;
Contrast Rosetta (DE3) all can not expand band.
In the integron of screening, all can expand bright band with these two pairs of primers, size is correct, for required.Obtain intestinal bacteria transformation host Rosetta (DE3):: ECack.
(2) STH is at Rosetta (DE3):: the expression in ECack
The recombinant plasmid pTrc99a-STH Calcium Chloride Method of aforementioned structure is transformed above-mentioned intestinal bacteria transformation host Rosetta (DE3):: ECack, obtains bacterial classification Rosetta (DE3):: ECack/pTrc99a-STH.
(3) pET24a-STH-opt is transformed Rosetta (DE3):: ECack, obtains Rosetta (DE3):: ECack/pET24a-STH-opt; Transform Rosetta (DE3):: gshB-ack obtains Rosetta (DE3):: gshB-ack/pET24a-STH-opt.
4, bacterial strain conversion capability
Bacterial classification Rosetta (DE3) obtained above/pTrc99a-STH/pMWK-gshB-ack, Rosetta (DE3):: ECack/pTrc99a-STH, Rosetta (DE3):: gshB-ack/pTrc99a-STH, Rosetta (DE3):: ECack/pET24a-STH-opt and Rosetta (DE3):: the conversion capability test of gshB-ack/pET24a-STH-opt.
Fermentation and bacterial cell disruption are with embodiment 1-12.
Conversion condition: reaction final volume 50ml (phosphoric acid buffer 0.1mM, pH7.2): Cys: 80mM, glycine: 120mM, L-sodium: 120mM, seven aqueous magnesium chlorides: 40mM, acetylphosphate dilithium salt: 120mM, ATP:1mM, bacterial cell disruption liquid: 20%V/V.First successively chemical reagent is added phosphoric acid buffer by above-mentioned formula, then adjust pH7.2, add bacterial cell disruption liquid, load 250ml shaking flask, seal after logical nitrogen, 30 DEG C, 150rpm reacts, and gets 500 μ l samples and adds in EP pipe, then add 500 μ l10% hydrochloric acid stopped reactions.Result is as following table 4.
Table 4
| 1h | 2h | 3h | 4h | |
| Rosetta(DE3)/pTrc99a-STH/pMWK-gshB-ack | 14.9g/L | 20.4g/L | 20.8g/L | 21.1g/L |
| Rosetta(DE3)::ECack/pTrc99a-STH | 14.2g/L | 15.8g/L | 18.4g/L | 18.2g/L |
| Rosetta(DE3)::gshB-ack/pTrc99a-STH | 12.2g/L | 15.8g/L | 17.8g/L | 18.2g/L |
| Rosetta(DE3)::ECack/pET24a-STH-opt | 8.7g/L | 15.1g/L | 17.6g/L | 18.1g/L |
| Rosetta(DE3)::gshB-ack/pET24a-STH-opt | 8.4g/L | 14.2g/L | 17.1g/L | 18.5g/L |
As can be seen here, Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack has the highest transformation efficiency.The bacterial strain of other recombinant expressed STH and ack also has relatively high transformation efficiency.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (13)
1. produce a method for gsh, it is characterized in that, described method comprises:
(1) process LAN gsh bifunctional enzyme and E.C. 2.7.2.1 in Bacillus coli cells, obtain recombinant expressed cell; With
(2) cell of broken (1), by cytoclasis product and Cys, glycine, Pidolidone or its salt, inorganic salt and ATP hybrid reaction, produces gsh.
2. the method for claim 1, is characterized in that, in step (1), and also process LAN glutathione synthetase in Bacillus coli cells.
3. method as claimed in claim 1 or 2, it is characterized in that, in step (1), described gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene and/or glutathione synthetase-coding gene gshB are expressed in same Bacillus coli cells culture systems; Or be expressed in different intestinal bacteria culture systems.
4. method as claimed in claim 1 or 2, it is characterized in that, step (1) comprising:
A () provides a kind of recombinant Bacillus coli cells, comprise the recombinant expression cassettes of following gene in described recombinant Bacillus coli cells: gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene, and selectivity contains glutathione synthetase-coding gene gshB; Or
Recombinant Bacillus coli cells is provided, comprises: a kind of comprise the recombinant expression cassettes of gsh bifunctional enzyme encoding gene Bacillus coli cells and a kind ofly comprise the Bacillus coli cells of the recombinant expression cassettes of acetate kinase-encoding gene and/or a kind of Bacillus coli cells comprising the recombinant expression cassettes of glutathione synthetase-coding gene gshB; With
B () cultivates the recombinant Bacillus coli cells of (a), thus process LAN gsh bifunctional enzyme and E.C. 2.7.2.1.
5. the method for claim 1, is characterized in that, described intestinal bacteria are Rosetta (DE3).
6. the method for claim 1, is characterized in that, described gsh bifunctional enzyme is the gsh bifunctional enzyme deriving from thermophilus streptococcus (Streptococcus thermophilus);
Described E.C. 2.7.2.1 derives from Lactobacillus sanfrancisco (Lactobacillus sanfranciscensis); Or derive from the E.C. 2.7.2.1 of intestinal bacteria (Escherichia coli).
7. the method for claim 1, is characterized in that, in step (2), described inorganic salt comprise: magnesium salts, acetyl phosphate.
8. the method for claim 1, is characterized in that, in step (2), reaction system comprises: Cys: 80 ± 40mM; Glycine: 120 ± 40mM; L-sodium: 120 ± 40mM; Seven aqueous magnesium chlorides: 40 ± 30mM; Acetylphosphate dilithium salt: 120 ± 40mM; ATP:1 ± 0.5mM; Cytoclasis product: 10 ~ 30%V/V.
9. a recombinant expression vector, is characterized in that, described recombinant expression vector comprises the recombinant expression cassettes of following gene: gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene and/or glutathione synthetase-coding gene gshB.
10. a Bacillus coli cells for restructuring, it is characterized in that, described Bacillus coli cells comprises expression vector according to claim 9, or
Be integrated with the recombinant expression cassettes of following gene in the genome of described Bacillus coli cells: gsh bifunctional enzyme encoding gene and acetate kinase-encoding gene, and selectivity contains glutathione synthetase-coding gene gshB; Preferably, described intestinal bacteria are Rosetta (DE3).
The purposes of 11. recombinant expression vectors according to claim 9 or recombinant Bacillus coli cells according to claim 10, is characterized in that, is gsh for transforming Cys, Pidolidone and glycine.
12. 1 kinds, for the production of the test kit of gsh, is characterized in that, described test kit comprises: the Bacillus coli cells of recombinant expression vector according to claim 8 or restructuring according to claim 10.
13., as claimed in claim 12 for the production of the test kit of gsh, is characterized in that, also comprise in described test kit: Cys, glycine, Pidolidone or its salt, inorganic salt and ATP; Preferably, described inorganic salt comprise: seven aqueous magnesium chlorides, acetylphosphate dilithium salt.
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