CN104611396B - A method of producing glutathione - Google Patents
A method of producing glutathione Download PDFInfo
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Abstract
The present invention relates to a kind of methods of improved production glutathione.The present inventor recombinantly expresses host by screening and the suitable enzyme of selection is overexpressed, obtain can Efficient Conversion L cysteines, L glutamic acid and glycine be glutathione recombinant bacterial strain, the yield of GSH can be effectively improved using the bacterial strain.50% or more glutathione yield can be improved in the method for the present invention.
Description
Technical field
The invention belongs to biotechnologies；More particularly it relates to a kind of side of improved production glutathione
Method.
Background technology
Glutathione (Glutathione, GSH) is a kind of drug of important regulation of physiological functions, answering clinically
With more and more extensively, it has also become important one of the drug with adjusting immune function of human body and anticancer adjuvant of medicine.The antioxygen of GSH
The property changed makes its application in the food industry receive people's concern again, fresh-keeping and improve product special flavour, improve and produce in food storage
Effect in terms of product nutritive value is increasingly expected.Glutathione be by Pidolidone, L-cysteine, glycine condensation and
At a kind of three peptides of bioactivity containing 2 glutamyls of γ and sulfydryl, synthesis, amino acid in protein and DNA
Transhipment, cell the important biological phenomena such as protection in play direct or indirect effect.It is distributed mainly on animal, plants
In object, microbial cell, it is widely used in many fields such as clinical medicine, sports health, food processing.Therefore, GSH has become
The hot spot studied and explored for scientists from all over the world.The method of production glutathione mainly has extraction, chemistry to close both at home and abroad at present
Cheng Fa, fermentation method, enzyme process.Since there is fermentation method production GSH strain to be easy to culture, the convenient cheap, reaction step of raw material sources
Simply, at low cost, the advantages that transformation efficiency is high, throughput rate is fast, it has also become the main method of current production glutathione.
A series of bacterial strain that production GSH are had existed in currently available technology, can usually give birth under very wide condition of culture
GSH is grown and accumulates, but yield is generally very low.There are the project urgently captured on many theory and technologies, such as superior strain at present
Selection and breeding and condition of culture optimization etc..Therefore, there is an urgent need for further study biosynthesis or the life of bio-conjugate chemistry method for this field
Produce the new method of glutathione.
Invention content
The purpose of the present invention is to provide a kind of methods of improved production glutathione.
In the first aspect of the present invention, a kind of method producing glutathione is provided, the method includes：
(1) (external source) glutathione bifunctional enzyme (STH) and acetokinase are overexpressed in Bacillus coli cells
(ack), recombinant expression cell is obtained；With
(2) it is crushed the cell of (1), (such as with L-cysteine, glycine, Pidolidone or its salt by clasmatosis product
Sodium salt), inorganic salts and ATP hybrid reactions, produce glutathione.
In a preference, in step (1), (external source) glutathione synthesis is overexpressed also in Bacillus coli cells
Enzyme.
In another preferred example, in step (1), the glutathione bifunctional enzyme encoding gene and acetokinase coding
Gene and/or glutathione synthetase-coding gene gshB are expressed in same Bacillus coli cells culture systems；Or it is expressed in
In different Escherichia coli culture systems.
In another preferred example, step (1) includes：
(a) a kind of recombinant Bacillus coli cells are provided, the recombination of following gene is included in the recombinant Bacillus coli cells
Expression cassette：Glutathione bifunctional enzyme encoding gene and acetate kinase-encoding gene, and selectivity contain glutathione synthesis
Enzyme coding gene gshB；Or
Recombinant Bacillus coli cells are provided, including：A kind of recombinant expression including glutathione bifunctional enzyme encoding gene
The Bacillus coli cells of box and a kind of Bacillus coli cells and/or one kind of the recombinant expression cassettes comprising acetate kinase-encoding gene
Include the Bacillus coli cells of the recombinant expression cassettes of glutathione synthetase-coding gene gshB；With
(b) recombinant Bacillus coli cells for cultivating (a), to be overexpressed glutathione bifunctional enzyme and acetokinase.
In another preferred example, the Escherichia coli are Rosetta (DE3).
In another preferred example, the glutathione bifunctional enzyme is derived from streptococcus thermophilus (Streptococcus
Thermophilus glutathione bifunctional enzyme) is (preferably, its gene order such as NCBI accession number：GU138096.1 or its
The sequence after codon optimization is carried out according to e. coli codon preferences)；
The acetokinase be derived from Lactobacillus sanfrancisco (Lactobacillus sanfranciscensis) (compared with
Goodly, gene order such as NCBI accession number：AB035799.1 or its according to e. coli codon preferences carry out codon
Sequence after optimization)；Or from Escherichia coli (Escherichia coli) (preferably, its gene order such as NCBI is logged in
Number：CP001509 or its according to e. coli codon preferences carry out codon optimization after sequence) acetokinase.
In another preferred example, in step (2), the inorganic salts include：Magnesium salts, acetyl phosphate.
In another preferred example, in step (2), reaction system includes：L-cysteine：80±40mM；Glycine：120
±40mM；L-sodium：120±40mM；Seven aqueous magnesium chlorides：40±30mM；Acetyl phosphate dilithium salt：120±40mM；ATP：
1±0.5mM；Clasmatosis product：10~30%V/V.
In another preferred example, the clasmatosis product refers to：After the recombinant expression cellular products centrifugation of step (1),
Breakdown products after being resuspended with 4 times of volume phosphate buffers.
In another preferred example, the temperature of reaction is 30 ± 5 DEG C；Preferably 30 ± 2 DEG C.
In another preferred example, the pH of reaction is 7.0 ± 0.5；Preferably 7.0 ± 0.2.
In another aspect of this invention, the recombinant expression carrier provided described in a kind of recombinant expression carrier includes following gene
Recombinant expression cassettes：Glutathione bifunctional enzyme encoding gene and acetate kinase-encoding gene and/or glutathione synthetase are compiled
Code gene gshB.
In another aspect of this invention, a kind of Bacillus coli cells of recombination are provided, the Bacillus coli cells include
The expression vector, or
The recombinant expression cassettes of following gene are integrated in the genome of the Bacillus coli cells：Glutathione bifunctional enzyme
(STH) encoding gene and acetokinase (ack) encoding gene, and selectivity contain glutathione synthetase-coding gene
gshB；Preferably, the Escherichia coli are Rosetta (DE3).
In another aspect of this invention, the use of the recombinant expression carrier or the recombinant Bacillus coli cells is provided
On the way, it is glutathione for converting L-cysteine, Pidolidone and glycine.
In another aspect of this invention, a kind of kit for producing glutathione is provided, the kit includes：
The Bacillus coli cells of the recombinant expression carrier or the recombination.
In a preference, the described kit for producing glutathione further includes in the kit：L- half
Cystine, glycine, Pidolidone or its salt (such as sodium salt), inorganic salts and ATP；Preferably, the inorganic salts include：Seven water
Magnesium chloride, acetyl phosphate dilithium salt.
The other aspects of the present invention are apparent to those skilled in the art due to this disclosure
's.
Description of the drawings
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, 1-3：
The full cell of BL21 (DE3)/pET24a-SAG, supernatant precipitation；10-12：The full cell of BL21 (DE3)/pET24a-STH, on
Cleer and peaceful precipitation；4-6：The full cell of Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack, supernatant precipitation；7-9：
The full cell of Rosetta (DE3)/pTrc99a-STH, supernatant precipitation；M is protein molecular weight standard (KDa).
The protein electrophoresis of Figure 18, BL21 (DE3)/pET24a-gshA and BL21 (DE3)/pET24a-gshB.
1：The precipitation of BL21 (DE3)/pET28b-gshA；
2：The supernatant of BL21 (DE3)/pET28b-gshA；
3：The precipitation of BL21 (DE3)/pET24a-gshB；
4：The supernatant of BL21 (DE3)/pET24a-gshB.
Specific implementation mode
In order to improve glutathione (Glutathione, GSH) yield, the present inventor passes through in-depth study, passes through screening
Recombinant expression host and the suitable enzyme of selection are overexpressed, obtain can Efficient Conversion L-cysteine, Pidolidone and
Glycine is the recombinant bacterial strain of glutathione, and the yield of GSH can be effectively improved using the bacterial strain.The method of the present invention can
Improve 50% or more glutathione yield.
As used herein, as used herein, " expression cassette " refer to include expression desired polypeptides (present invention in be
Glutathione bifunctional enzyme (STH), acetokinase (ack) and/or glutathione synthetase gshB) needed for all necessary components
Gene expression system, usually it include following elements：Promoter, the gene order for encoding polypeptide, terminator；It is additionally optional
Selecting property includes signal coding sequence etc.；These elements are operatively connected.
Therefore, the present invention provides a kind of construction, the construction includes：STH, ack and include selectively
The expression cassette of gshB.The expression cassette has all elements (including promoter, coding DNA and end needed for gene expression
Only son etc.), so as to completely give expression to corresponding albumen.
In general, the construction is located on expression vector.Therefore, the invention also includes a kind of carriers, it contains described
Construction.The expression vector usually also contains replication orgin and/or marker gene etc..Those skilled in the art is known
Method can be used to build the required expression vector of the present invention.These methods include recombinant DNA technology in vi, DNA synthetic technologys,
In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to close
At.Expression vector further includes the ribosome bind site and transcription terminator of translation initiation.
In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion
The phenotypic character of host cell, such as apramycin (apr) resistance, Amp resistances.
The carrier for including above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence can be used for turning
Change host appropriate.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell, such as calcium chloride
Method, electrotransformation.
Cell of the present invention for recombinant expression is Escherichia coli；Preferably Rosetta (DE3).The present inventor exceeds
Expect ground to find, is overexpressed glutathione bifunctional enzyme (STH), acetokinase (ack) and/or glutathione synthetase
Rosetta (DE3) bacterial strain can significantly improve the yield of GSH for producing GSH after cracking.
The present invention also provides a kind of method producing glutathione, the method includes：(1) in Bacillus coli cells
It is overexpressed (external source) glutathione bifunctional enzyme (STH) and acetokinase (ack), obtains recombinant expression cell；(2) it breaks
The cell of broken (1), by clasmatosis product and L-cysteine, glycine, Pidolidone or its salt (such as sodium salt), inorganic salts and
ATP hybrid reactions produce glutathione.
In the early-stage study of the present inventor, may have for the production of glutathione using Bacillus coli expression is a variety of
The enzyme of profit finally found that the cellular lysate liquid for being overexpressed glutathione bifunctional enzyme (STH) and acetokinase (ack) is conducive to
The raising of yield.
In addition, in early-stage study, the present inventor has also attempted using a variety of Escherichia coli (such as JM109, Origami
(DE3), DH5 α, BL21 (DE3), Rosetta (DE3) etc.) recombinantly expressed, finally found that using Rosetta (DE3) come into
Row recombinant expression can generate unexpected technique effect.
As the preferred embodiment of the present invention, the glutathione bifunctional enzyme derives from streptococcus thermophilus
(Streptococcus thermophilus)；The acetokinase derives from Lactobacillus sanfrancisco (Lactobacillus
sanfranciscensis)；Or derive from Escherichia coli (Escherichia coli).It should be understood that retaining natural egg
The protein variant of white essentially identical function, bioactive fragment are also included in the present invention.
The invention further relates to the kit for producing glutathione, the kit includes：For express STH and
The recombinant expression carrier of ack and selective expression gshB；Or the kit includes that can be overexpressed STH and ack
And the Bacillus coli cells or product of cell lysis of selective expression gshB.The recombinant expression carrier or cell or cell
Pyrolysis product is placed in container appropriate.
Further include producing glutathione for subsequent chemical reaction as the preferred embodiment of the present invention, in the kit
Other chemical compositions, including but not limited to：L-cysteine, glycine, Pidolidone or its salt (such as sodium salt), inorganic salts and
ATP；Preferably, the inorganic salts include：Seven aqueous magnesium chlorides, acetyl phosphate dilithium salt.
As the preferred embodiment of the present invention, further includes operation instructions in the kit, illustrate various chemical reagent
Or expression vector or the concentration of cell or product of cell lysis, usage and dosage.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brookers etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Strain is built
Main agents：Restriction enzyme and Taq archaeal dna polymerases (fermentas), T4DNA ligases, Klenow pieces
Section, alkaline phosphatase CIAP and protein molecular weight standard (TAKARA), KOD neo plusDNA polymerases (TOYOBO), matter
Grain extraction agent box and a small amount of plastic recovery kits (Axygen), antibiotic (Shanghai life work).Primer synthesis, full genome synthesis and
All commission Nanjing Genscript Biotechnology Co., Ltd. completes subclone.
Embodiment 1, production glutathione (GSH) single expression of enzymes strain structure
1, the structure of pET24a-SAG
According to the gene order of the sources the Streptococcus agalactiae glutathione bifunctional enzyme SAG reported
(NCBI accession number：AE009948.1), full genome synthesizes the sequence, and restriction enzyme site NdeI and HindIII, subclone are designed in both ends
Corresponding site on to carrier pET24a (being purchased from Novagen), obtains recombinant plasmid pET24a-SAG, plasmid map is shown in Fig. 1.
The recombinant plasmid pET24a-SAG built Calcium Chloride Methods are converted into Bacillus coli expression host BL21 (DE3),
Obtain BL21 (DE3)/pET24a-SAG.
2, the structure of pET24a-STH and expression
According to having reported the double work(of the sources Streptococcus thermophilus strain SIIM B218 glutathione
Gene order (the NCBI accession number of energy enzyme STH：GU138096.1), full genome synthesizes the sequence, and restriction enzyme site is designed at both ends
EcoRI and XhoI is subcloned into corresponding site on carrier pET24a, obtains recombinant plasmid pET24a-STH, and plasmid map is shown in figure
2。
By the recombinant plasmid pET24a-STH built with Calcium Chloride Method convert Bacillus coli expression host BL21 (DE3) and
Rosetta (DE3) obtains BL21 (DE3)/pET24a-STH and Rosetta (DE3)/pET24a-STH.
3, the structure of pET28b-gshA
According to the source Escherichia coli BL21 (DE3) the Glutamate-cysteine ligase gene reported
Gene order (the NCBI accession number of gshA：AM946981.2), design primer is as follows：gshA-NcoI-F：
CATGCCATGGGAATCCCGGACGTATCACAGGC(SEQ ID NO:1), gshA-BamHI-R：
CGGGATCCTCAGGCGTGTTTTTCCAGCC(SEQ ID NO:2), using BL21 (DE3) genomic DNAs template amplification gshA
Segment.PCR reaction systems include：Each 0.3 μM of primer, template 200ng, 1X KOD neo plus buffer, 0.2mM dNTP,
1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR amplification condition is：94 DEG C of 2min, 98 DEG C of 10s,
55 DEG C of 30s, 68 DEG C of 45s repeat 30 cycles, 68 DEG C of 10min.PCR after reaction, is divided with agarose gel electrophoresis
Analysis, detects the specific band of an about 1.5kb or so, is recycled with plastic recovery kit, and NcoI and BamHI are in 37 DEG C of double enzymes
It cuts 3-6 hours, digestion system includes：42 μ L, 10 × Tango buffer5 μ L, NcoI1.5 μ L, BamHI1.5 μ L of PCR product.
Plastic recovery kit crosses column purification recycling.The expression vector pET28b (being purchased from Novagen) of recovery product and same digestion processing
It is stayed overnight in 16 DEG C of connections with T4DNA ligases, Transformed E .coli DH5 α competent cells obtain recombinant plasmid pET28b-
GshA, plasmid map are shown in Fig. 3, and BL21 (DE3) is converted after extraction and obtains BL21 (DE3)/pET28b-gshA.
4, the structure of pET24a-gshB
According to the base of Escherichia coli BL21 (DE3) the source Glutatione synthetase gene gshB reported
Because of sequence (NCBI accession number：AM946981.2), design primer is as follows：gshB-NdeI-F：
GGAATTCCATATGatcaagctcggcatcgt(SEQ ID NO:3), gshB-BamHI-R：
CGGGATCCttactgctgctgtaaacgtg(SEQ ID NO:4), using BL21 (DE3) genomic DNAs template amplification gshB
Segment.PCR reaction systems include：Each 0.3 μM of primer, template 200ng, 1 × KODneo plus buffer, 0.2mM dNTP,
1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR amplification condition is：94 DEG C of 2min, 98 DEG C of 10s,
55 DEG C of 30s, 68 DEG C of 30s repeat 30 cycles, 68 DEG C of 10min.PCR after reaction, is divided with agarose gel electrophoresis
Analysis, detects the specific band of an about 1kb or so, is recycled with plastic recovery kit, NdeI and BamHI are in 37 DEG C of double digestions
3-6 hours, digestion system included：37 μ L, 10 × Tango buffer10 μ L, NdeI1.5 μ L, BamHI1.5 μ L of PCR product.Glue
QIAquick Gel Extraction Kit crosses column purification recycling.The expression vector pET24a of recovery product and same digestion processing with T4DNA ligases in
Overnight, Transformed E .coli DH5 α competent cells obtain recombinant plasmid pET24a-gshB, and plasmid map is shown in Fig. 4 for 16 DEG C of connections,
BL21 (DE3) is converted after extraction obtains BL21 (DE3)/pET24a-gshB.
5, the protein electrophoresis of above-mentioned strain
The strain built is inoculated into respectively in the LB liquid medium containing 100 μ g/mL kanamycins, 37 DEG C of 220rpm
After overnight incubation, it is inoculated into fresh TB culture mediums by 1% (v/v) inoculum concentration and (is contained 100 μ g/mL kanamycins), 37 DEG C of 220rpm
Final concentration of 0.2mMIPTG, 28 DEG C of overnight inductions are added to OD600=5-6 in culture.
It is resuspended with 20ml0.1M PBS buffer solution (pH7.0) after taking the bacterium solution 10ml after induction to collect thalline, ultrasonication,
Be respectively set full cell liquid (1ml breaks cytosol), supernatant (1ml break cytosol centrifugation after take supernatant), precipitated liquid (1ml break cytosol from
Abandon supernatant after the heart, 1ml buffer solutions be added, precipitation is resuspended) three parts of samples, by each 20 μ l of electrophoresis Sample and 5 μ l5 × sample buffer
Mixing is boiled 5 minutes, and 10 μ L of supernatant is taken to carry out SDS-PAGE analyses after centrifugation.Using 5% concentration glue and 12% separation gel, electrophoresis knot
Gel is dyed with coomassie brilliant blue R250 after beam.As a result such as Figure 17-18.
6, the structure of pTrc99a-STH and expression
With EcoRI and XhoI in 37 DEG C of double digestions 3-6 hours, digestion system includes recombinant plasmid pET24a-STH：Plasmid
37 μ L, 10 × Tango buffer10 μ L, EcoRI1.5 μ L, XhoI1.5 μ L.Gel electrophoresis plastic recovery kit recycles 2.2kb
STH segments.
Carrier pTrc99a (be purchased from Pharmacia) is with EcoRI and SalI in 37 DEG C of double digestions 3-6 hours, digestion system packet
It includes：15 μ L, 10 × Buffer O5 μ L, EcoRI1.5 μ L, SalI1.5 μ L of plasmid, moisturizing to 50 μ L of total system.Glue reclaim reagent
Box crosses column and returns pTrc99a endonuclease bamhis.
It is connected, above-mentioned two segment is stayed overnight with T4DNA ligases in 16 DEG C of connections, even using XhoI with SalI isocaudarners
It practices midwifery object Transformed E .coli DH5 α competent cells, is coated with Amp tablets.
Single bacterium colony is chosen from conversion tablet and connects LB Tube propagations, plasmid is extracted with plasmid extraction kit, obtains recombination matter
Grain pTrc99a-STH, plasmid map are shown in Fig. 5.EcoRI and HindIII double digestions are used to verify later, if 2.2kb segments can be cut out
It is then correct.
The recombinant plasmid pTrc99a-STH built is converted into Bacillus coli expression host JM109 with Calcium Chloride Method respectively
(giving birth to work purchased from Shanghai), Origami (DE3) (being purchased from Novagen) and Rosetta (DE3) (being purchased from Novagen) obtain JM109/
PTrc99a-STH, Origami (DE3)/pTrc99a-STH and Rosetta (DE3)/pTrc99a-STH.
7, the structure of pSUGAP-STH and expression
Plasmid pSU2718 (Martinez, E. etc., Gene1988,68,159) is with SacI and Sma I in 37 DEG C of double digestion matter
Grain 3-6h, digestion system are：37 μ L, 10 × Tango buffer10 μ L, SacI1.5 μ L, SmaI1.5 μ L of plasmid, glue recycling examination
Agent box recycles the pSU2718 segments of 2.3kb.
Design primer is as follows：ecgapup：GGTACCGAGCTCGAGGCGAGTCAGTCGCGTAATGC(SEQ ID NO:
5)；ecgapdn：TTTCCCGGGTTAATTAAGATCTATATTCCACCAGCTATTTGTTAG(SEQ ID NO:6), pass through PCR
The 0.2kb segments comprising GAP promoters are isolated from genome of E.coli, PCR reaction systems include：Each 0.3 μM of primer,
Template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO4, KOD neoplus1U, benefit ddH2O
To 50 μ L of total system.Temperature condition is：94℃2min；98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 20s repeat 30 cycles；68℃
10min.PCR after reaction, agarose gel electrophoresis identification, plastic recovery kit recycle GAP segments, with SacI and SmaI in
37 DEG C of double digestion 3-6h, digestion system are：37 μ L, 10 × Tango buffer10 μ L, SacI1.5 μ L, SmaI1.5 μ of GAP segments
L, plastic recovery kit cross the pSU2718 segments for the 2.3kb that after column recycling and above-mentioned recycling obtains under the action of T4 ligases
Overnight in 16 DEG C of water-bath connections, Transformed E .coli DH5 α competent cells are coated with Cm tablets, 37 DEG C of overnight incubations.Select conversion
Son connects LB Tube propagations and stays overnight, and with kit extracting plasmid verification, obtains pSUGAP plasmids, plasmid map is shown in Fig. 6.
PET24a-STH does substep digestion in 37 DEG C with NheI and XhoI, and glue recycles 2.3kb or so STH segments, pSUGAP
With XbaI and SalI (2 × Buffer Tango) in 37 DEG C of double digestion 3-6h, crosses column purification and recycle pSUGAP carrier segments.This two
A segment is stayed overnight with T4DNA ligases in 16 DEG C of connections, is converted DH5 α competent cells, is coated with Cm tablets, 37 DEG C of overnight incubations.
Single bacterium colony is chosen from conversion tablet and connects LB Tube propagations, and upgrading grain is verified with BamHI single endonuclease digestions, and correct recombinant clone can be cut
Go out 1kb or so segments, plasmid map is shown in Fig. 7.
The recombinant plasmid pSUGAP-STH built Calcium Chloride Methods are converted Bacillus coli expression host BL21 (DE3) to obtain
To BL21 (DE3)/pSUGAP-STH.
8, the structure of pET32amal-STH and expression
PMal-c2x (being purchased from New England Biolabs) is with NdeI and HindIII in 37 DEG C of double digestion 3-6h, digestion
System is：42 μ L, 10 × Buffer R5 μ L, NdeI1.5 μ L, HindIII1.5 μ L of plasmid.Agarose gel electrophoresis, which detects, to be used in combination
Plastic recovery kit recycles 1.2kb mal segments, is used with pET32a (being purchased from novagen) segment of same double digestion processing
T4DNA ligases overnight, convert DH5 α competent cells in 16 DEG C of connections, are coated with Amp tablets, 37 DEG C of overnight incubations.From conversion
Single bacterium colony is chosen on tablet and connects LB Tube propagations, and upgrading grain is verified with NdeI/HindIII double digestions, and correct recombinant clone can be with
1.2kb or so segments are cut out, pET32amal plasmids are obtained, plasmid map is shown in Fig. 8.
PET24a-STH with EcoRI and XhoI, stay overnight by the double digestion in 37 DEG C of water-baths, and digestion system is：Plasmid 37 μ L, 10
× Buffer Tango10 μ L, EcoRI1.5 μ L, XhoI1.5 μ L.Glue recycles at 2.2kb or so STH segments, with same double digestion
The pET32amal segments of reason are stayed overnight with T4DNA ligases in 16 DEG C of connections, are converted DH5 α competent cells, are coated with Amp tablets,
37 DEG C of overnight incubations.Single bacterium colony is chosen from conversion tablet and connects LB Tube propagations, and upgrading grain is verified with EcoRI/XhoI double digestions, just
True recombinant clone can cut out 2.2kb or so large fragments.Plasmid map is shown in Fig. 9.
The recombinant plasmid pET32amal-STH built is converted into Bacillus coli expression host BL21 with Calcium Chloride Method respectively
(DE3), Origami (DE3) and Rosetta (DE3), obtains BL21 (DE3)/pET32amal-STH, and Origami (DE3)/
PET32amal-STH and Rosetta (DE3)/pET32amal-STH.
9, the structure of pET24a-ack and expression
According to the gene sequence of the sources the Lactobacillus sanfranciscensis Acetokinase gene ack reported
Arrange (NCBI accession number：AB035799.1), full genome synthesizes the sequence, and restriction enzyme site NdeI and BamHI, subclone are designed in both ends
Corresponding site on to carrier pET24a, obtains recombinant plasmid pET24a-ack, and plasmid map is shown in Figure 10.
The recombinant plasmid Calcium Chloride Method built conversion Bacillus coli expression host BL21 (DE3) is expressed, is obtained
Acetokinase expresses strain BL21 (DE3)/pET24a-ack.
10, the structure of pET24a-ECack and expression
According to the gene order of Escherichia coli BL21 (DE3) the source Acetokinase gene ECack reported
(NCBI accession number：CP001509.3), design primer is as follows：ECack-NdeI-F：
GGAATTCCATATGTCGAGTAAGTTAGTAC(SEQ ID NO:7), ECack-BamHI-R：
CGGGATCCTCAGGCAGTCAGGCGGCT(SEQ ID NO:8), using BL21 (DE3) genomic DNAs template amplification ECack pieces
Section.PCR reaction systems include：Each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM dNTP,
1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR amplification condition is：94 DEG C of 2min, 98 DEG C of 10s,
55 DEG C of 30s, 68 DEG C of 45s repeat 30 cycles, 68 DEG C of 10min.PCR after reaction, is divided with agarose gel electrophoresis
Analysis, detects the specific band of an about 1.2kb or so, is recycled with plastic recovery kit, and NdeI and BamHI are in 37 DEG C of double enzymes
It cuts 3-6 hours, digestion system includes：37 μ L, 10 × Buffer Tango10 μ L, NdeI1.5 μ L, BamHI1.5 μ L of PCR product.
Plastic recovery kit crosses column purification recycling.
Recovery product is connect overnight with the expression vector pET24a that same digestion is handled with T4DNA ligases in 16 DEG C, is turned
Change E.coli DH5 α competent cells, obtain recombinant plasmid pET24a-ECack, plasmid map is shown in Figure 11, is converted after extraction
BL21 (DE3) obtains BL21 (DE3)/pET24a-ECack.
11, STH codon optimizations are expressed
To the gene order (sources Streptococcus thermophilus strain SIIM B218) of STH, according to
After the codon-bias of Escherichia coli carries out codon optimization, recombines to obtain STH-opt, be then integrated into pET24a matter
Grain conversion BL21 (DE3) obtains BL21 (DE3)/pET24a-STH-opt.
Following (the SEQ ID NO of STH-opt sequences:9)：
atgaccctgaaccaactgctgcaaaaactggaagcgacctcgccgatcctgcaagccaacttcggtattgaacgtga
aagcctgcgtgtcgatcgccagggtcaactggtgcataccccgcacccgagttgcctgggtgcccgttccttccatc
cgtatatccagaccgatttctgtgaatttcaaatggaactgatcacgccggttgcgaaaagcaccacggaagcccgt
cgctttctgggcgccatcaccgatgtcgcaggtcgctcaattgctacggacgaagtgctgtggccgctgagcatgcc
gccgcgtctgaaagcagaagaaattcaggtggctcaactggaaaacgatttcgaacgtcattatcgcaattacctgg
cagaaaaatatggcaccaagctgcaggctattagcggtatccactacaacatggaactgggcaaagatctggttgaa
gcactgttccaggaatctggtcaaaccgacatgatcgcttttaaaaacgcgctgtatctgaagctggcgcagaatta
tctgcgttaccgctgggtgatcacgtacctgtttggtgccagtccgattgcagaacagggctttttcgatcaagaag
tgccggaaccggttcgtagcttccgcaactctgaccatggttatgtgaacaaagaagaaatccaggtgtctttcgtt
tccctggaagattatgtttccgcgattgaaacctacatcgaacagggcgacctgaacgcagaaaaggaattttacag
tgctgttcgtttccgcggtcaaaaagtcaatcgctccttcctggataagggcattacctacctggaatttcgcaact
tcgacctgaatccgtttgaacgtattggtatctcacagaccacgatggatacggtccacctgctgatcctggccttt
ctgtggctggattcgccggaaaacgtggaccaggctctggcgcaaggccatgcgctgaatgaaaaaattgcgctgag
tcacccgctggaaccgctgccgtccgaagccaagacccaggatatcgtgacggccctggaccagctggttcaacatt
tcggcctgggtgattatcaccaggacctggtcaaacaagtgaaggcggcctttgcagatccgaaccagaccctgagc
gctcaactgctgccgtacattaaggataagtctctggctgaatttgcgctgaataaagccctggcatatcatgatta
cgactggaccgcccactatgcactgaaaggttacgaagaaatggaactgagcacgcagatgctgctgtttgatgcga
ttcaaaaaggcatccatttcgaaattctggatgaacaggaccaatttctgaagctgtggcatcaggatcacgttgaa
tatgtcaaaaacggtaatatgacctctaaggacaactacgtggttccgctggctatggcgaataaaaccgttacgaa
aaagattctggccgatgcaggctttccggtcccgtcaggtgacgaatttacctcgctggaagaaggcctggcctatt
acccgctgatcaaagataagcagattgtcgtgaaaccgaagtcaacgaactttggcctgggtattagcatcttccag
gaaccggcgtctctggataattatcaaaaagccctggaaattgctttcgcggaagacacctcggttctggtcgaaga
atttatcccgggtacggaataccgtttctttattctggatggccgctgcgaagcggtcctgctgcgtgtggcagcta
atgttatcggcgatggtaaacataccattcgcgaactggtggctcagaagaacgcgaatccgctgcgtggtcgtgat
caccgtagcccgctggaaattatcgaactgggcgacatcgaacagctgatgctggcacagcaaggttataccccgga
tgacattctgccggaaggcaaaaaggttaacctgcgtcgcaactcaaatatctcgacgggcggtgatagtattgaca
tcaccgaaacgatggatagctcttatcaggaactggccgcggcaatggccaccagcatgggtgcatgggcatgtggt
gtggatctgattatcccggacgaaacccagatcgccacgaaagaaaatccgcattgcacctgtattgaactgaactt
taatccgagcatgtatatgcacacgtactgtgcggaaggcccgggtcaggcgatcacgacgaagattctggataaac
tgttcccggaaattgtggcaggtcaaacgtaa
12, the GSH determinations of yield that the different strain of aforementioned structure is shared with BL21 (DE3)/pET24a-ack
The fermentation process of various strains：LB culture mediums (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L,
pH7.2)；TB culture mediums (peptone 12g/L, yeast extract 24g/L, glycerine 5g/L, KH2PO42.13g/L K2HPO4·
3H2O16.43g/L, pH7.0-7.5)；121 DEG C of autoclave sterilizations 20 minutes.Shake flask culture conditions：By the strain built point
It is not inoculated into the LB liquid medium for respectively corresponding to antibiotic containing 100 μ g/mL, after 37 DEG C of 220rpm overnight incubations, is connect by 1%
Kind amount is inoculated into fresh TB culture mediums and (respectively corresponds to antibiotic containing 100 μ g/mL), and 37 DEG C of 220rpm are cultivated to OD600=5-6,
Final concentration of 0.2mM IPTG, 28 DEG C of overnight inductions are added.
Bacterial cell disruption liquid processing method：Fermentation centrifugation obtains the phosphate buffer (0.1mM, pH7.2) of 4 times of volumes of thalline
It is resuspended, then ultrasonication.
Produce GSH reaction systems：React final volume 5ml (phosphate buffer 0.1mM, pH6.8)：L-cysteine：80mM,
Glycine：120mM, L-sodium：120mM, seven aqueous magnesium chlorides：40mM, acetyl phosphate dilithium salt：120mM, ATP：1mM, second
Acid kinase thalline (BL21 (DE3)/pET24a-ack) is crushed liquid 0.4% (V/V), the broken liquid of each recombinant bacterium of aforementioned structure：
(wherein BL21 (DE3)/pET28b-gshA and BL21 (DE3)/pET24a-gshB are according to different proportion mixed determining enzyme by 10%V/V
Living, total dosage of the two is 10% (V/V)).Phosphate buffer first is added in chemical reagent successively by above-mentioned formula, is then adjusted
Bacterial cell disruption liquid is added in pH6.8, is packed into test tube, is sealed after logical nitrogen, 30 DEG C, 150rpm reactions take 500 after reacting 30 minutes
μ l samples are added in EP pipes, add 500 μ l10% hydrochloric acid stopped reactions.
The yield data measured such as table 1.
Table 1
| Strain | 30min GSH contents |
| 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 |
By above-mentioned data it is found that the production that Rosetta (DE3)/pTrc99a-STH and BL21 (DE3)/pET24a-ack is shared
Measure highest.
13, the comparison of two kinds of ACK
By the conversion of Rosetta (DE3)/pTrc99a-STH, compare influence situations of the separate sources ack to conversion
(BL21 (DE3)/pET24a-ack and BL21 (DE3)/pET24a-ECack (large intestine source ack).
Fermentation and bacterial cell disruption technique are the same as embodiment 1-12
Comparison condition：React final volume 50ml (phosphate buffer 0.1mM, pH6.8)：L-cysteine：80mM, sweet ammonia
Acid：120mM, L-sodium：120mM, seven aqueous magnesium chlorides：40mM, acetyl phosphate dilithium salt：120mM, ATP：1mM, different second
Acid kinase bacterial cell disruption liquid 0.4%V/V, Rosetta (DE3)/pTrc99a-STH bacterial cell disruption liquid：10%V/V.First match by above-mentioned
Phosphate buffer is added in chemical reagent by Fang Yici, then adjusts pH6.8, and bacterial cell disruption liquid is added, and is packed into 250ml shaking flasks, leads to nitrogen
It is sealed after gas, 30 DEG C, 150rpm reactions.After reaction 30 minutes, takes 500 μ l samples to be added in EP pipes, add 500 μ l10% salt
Sour stopped reaction.
As a result such as table 2.
Table 2
| 0.5h GSH yield | |
| BL21(DE3)/pET24a-ack | 8.26g/L |
| BL21(DE3)/pET24a-ECack | 8.92g/L |
By comparison, the inventors discovered that two kinds of kinases biomass using seldom in the case of can be turned well
Change.
14, Rosetta (DE3)/pTrc99a-STH, BL21 (DE3)/pET24a-SAG and BL21 (DE3)/pET28b-
GshA and BL21 (DE3)/pET24a-gshB mixes the comparison of conversion production GSH with BL21 (DE3)/pET24a-ack
Zymotechnique and bacterial cell disruption technique are the same as embodiment 1-12.
Conversion process：React final volume 50ml (phosphate buffer 0.1mM, pH7.2)：L-cysteine：80mM, sweet ammonia
Acid：120mM, L-sodium：120mM, seven aqueous magnesium chlorides：40mM, acetyl phosphate dilithium salt：120mM, ATP：1mM,
Rosetta (DE3)/pTrc99a-STH, BL21 (DE3)/pET24a-SAG and BL21 (DE3)/pET28b-gshA and BL21
(DE3) the bacterial cell disruption liquid of/pET24a-gshB：20%V/V (wherein BL21 (DE3)/pET28b-gshA and BL21 (DE3)/
For pET24a-gshB according to different proportion mixed determining enzyme activity, total dosage of the two is 20%V/V)；Acetokinase thalline (BL21
(DE3)/pET24a-ack) it is crushed liquid 0.4%V/V.Phosphate buffer first is added in chemical reagent successively by above-mentioned formula, then
PH7.2 is adjusted, bacterial cell disruption liquid is added, 250ml shaking flasks is packed into, is sealed after logical nitrogen, 30 DEG C, 150rpm reactions take 500 μ l samples
It is added in EP pipes, adds 500 μ l10% hydrochloric acid stopped reactions.
As a result such as table 3.
Table 3
| Strain | 1h GSH yield |
| 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 |
By above-mentioned data it is found that the production that Rosetta (DE3)/pTrc99a-STH and BL21 (DE3)/pET24a-ack is shared
Measure highest.
The structure for organizing synthase expression strain of embodiment 2, production GSH
1, the structure of STH and gshB-ack coexpressions strain
(1) structure of pMWK-gshB-ack
With plasmid pPIC3.5K (being purchased from Invitrogen) for masterplate, design primer is as follows：Kandn：CCAACCAATTAACC
AATTCTGATTAG(SEQ ID NO:10), Kanup：CCTGCAGGGGGGGGGGGAAAGCCACGTTGTGTC(SEQ ID NO:11),
PCR amplification about 0.9kb Kan segments.PCR reaction systems include：Each 0.3 μM of primer, template 50ng, 1 × KODneo plus
Buffer, 0.2mM dNTP, 1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.Temperature condition is：
94℃2min；98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 30s repeat 30 cycles；68℃10min.PCR after reaction, agarose
Gel electrophoresis identifies that plastic recovery kit recycles kan segments.Plasmid pMW118 (is purchased from Nippon Gene, Toyama, Japan)
With ApaLI and EcoO1109I in 37 DEG C of double digestion 3-6h, digestion system is：37 μ L, 10 × BufferTango10 μ L of plasmid,
ApaLI1.5 μ L, EcoO1109I1.5 μ L.Agarose gel electrophoresis detects blend compounds QIAquick Gel Extraction Kit and recycles 2.7kb segments, uses
3 ' female end of Klenow segments filling-in.Kan segments and pMW118 filling-in segment are stayed overnight with T4DNA ligases in 16 DEG C of connections, turned
Change DH5 α competent cells, is coated with Kan tablets, 37 DEG C of overnight incubations.Single bacterium colony, which is chosen, from conversion tablet utilizes KanF/M13F-
47 are bacterium colony PCR verifications (kanF：TCGGAATCGCAGACCGATAC(SEQ ID NO:12), M13F-47：
CGCCAGGGTTTTCCCAGTCACGAC(SEQ ID NO:13)), if 500bp segments can be amplified, be as connected into it is in the right direction,
PMWK is obtained, plasmid map is shown in Figure 12.
With BglII and BamHI in 37 DEG C of double digestions 3-6 hours, digestion system includes recombinant plasmid pET24a-ack：Plasmid
37 μ L, 10 × Tango buffer10 μ L, BglII1.5 μ L, BamHI1.5 μ L.Gel electrophoresis plastic recovery kit recycles 1.2kb
Ack segments.
With BamHI in 37 DEG C of single endonuclease digestions 3-6 hours, digestion system includes recombinant plasmid pET24a-gshB：30 μ L of plasmid,
10 × BamHI buffer5 μ L, BamHI1.5 μ L, moisturizing to 50 μ L of total system.Plastic recovery kit recycles 6.2kb carrier-pellets
Section.With alkaline phosphatase CIAP, the dephosphorylation in 37 DEG C of water-baths reacts 1 hour recovery product, and reaction system is：10×
44 μ L, CIAP1 μ L of Alkaline Phospharase buffer5 μ L, pET24a-gshB single endonuclease digestions piece.Plastic recovery kit mistake
Column purification dephosphorylation segment.
Above-mentioned two segment is stayed overnight with T4DNA ligases in 16 DEG C of connections, connection product Transformed E .coliDH5 α impressions
State cell is coated with Kan tablets.
Single bacterium colony is chosen from conversion tablet and connects LB Tube propagations, plasmid is extracted with plasmid extraction kit, obtains recombination matter
Grain pET24a-gshB-ack (Figure 13).BglII and BamHI double digestions are used to verify later, for just if it can cut out 2.3kb segments
Really.
GshB-ack segments are expanded by template design primer of recombinant plasmid pET24a-gshB-ack.Design of primers is as follows：
gshB-SalI-F：ACGCGTCGACATGATCAAGCTCGGCATC(SEQ ID NO:14), ack-BamHI-R：
CGGGATCCTTATTTATTTGCTAATGT(SEQ ID NO:15).PCR reaction systems include：Each 0.3 μM of primer, template
50ng, 1 × KOD neo plus buffer, 0.2mMdNTP, 1.5mM MgSO4, KOD neo plus1U mend ddH2O to total
50 μ L of system.PCR amplification condition is：94 DEG C of 2min, 98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 1min15s repeat 30 and recycle, 68 DEG C
10min.PCR after reaction, is analyzed with agarose gel electrophoresis, detects the special item of an about 2.3kb or so
Band is recycled with plastic recovery kit, and SalI and BamHI include in 37 DEG C of double digestions 3-6 hours, digestion system：42 μ of PCR product
L, 10 × Buffer BamHI5 μ L, SalI1.5 μ L, BamHI1.5 μ L.Plastic recovery kit crosses column purification recycling.
Plasmid pMWK equally uses SalI and BamHI in 37 DEG C of double digestions 3-6 hour, and plastic recovery kit recycles 3.6kb loads
Body segment.
The gshB-ack segments of recycling and pMWK segments are stayed overnight with T4DNA ligases in 16 DEG C of connections, connection product turns
Change E.coli DH5 α competent cells, is coated with Kan tablets.
Single bacterium colony is chosen from conversion tablet and connects LB Tube propagations, is extracted plasmid with plasmid extraction kit, is used SalI/
BamHI double digestions obtain recombinant plasmid pMWK-gshB-ack, plasmid map is shown in Figure 14 if it is correct that can cut out 2.3kb segments.
(2) structure of strain is co-expressed
By the plasmid pMWK-gshB-ack of above-mentioned structure with Calcium Chloride Method convert escherichia coli host Rosetta (DE3)/
PTrc99a-STH (embodiment 1-6 is obtained) competent cell, is coated with the LB tablets containing Cm/Amp/Kan, picking monoclonal is taken out
The correctness of upgrading grain verification conversion, obtains coexpression strain Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack.
2, expression of the STH in the escherichia coli host Rosetta (DE3) for integrating gshB-ack
The transformation of Bacillus coli expression host：GshB-ack is integrated into the sites yaiT of Rosetta (DE3) genome：
(1) structure of pTrc99a-gshB-ack-773
The aforementioned recombinant plasmid pET24a-gshB built NdeI/BamHI (2 × Buffer Tango) double digestions, glue
1kb or so gshB segments are recycled, corresponding site on carrier pET28b is cloned into, obtain recombinant plasmid pET28b-gshB.
PET28b-gshB NcoI/BamHI (1 × Buffer Tango) double digestions, glue recycle 1kb or so gshB segments,
It is cloned into corresponding site on carrier pTrc99a, obtains recombinant plasmid pTrc99a-gshB.
The aforementioned recombinant plasmid pET24a-ack built XbaI/SalI (2 × Buffer Tango) double digestions, glue return
1.2kb ack segments are received, corresponding site on carrier pTrc99a-gshB is cloned into, obtain recombinant plasmid pTrc99a-gshB-
ack。
Plasmid pIJ773 is (referring to 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 recycling two
While containing the apramycin resistance gene (1.5kb) in the sites FRT, by the pTc99a- of this recycling segment and same digestion processing
GshB-ack is stayed overnight with T4DNA ligases in 16 DEG C of connections, and connection product Transformed E .coliDH5 α competent cells are coated with Amp/
Apr tablets.Picking monoclonal extracts the correctness of plasmid verification conversion, obtains recombinant plasmid pTrc99a-gshB-ack-773,
Plasmid map is shown in Figure 15.
(2) using Rosetta (DE3) genomes as template amplification integration site yaiT gene upstream and downstream homologous sequences
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)
Using Rosetta (DE3) genomic DNAs template, two couples of primer yaiT-SwaI-F and yaiT-gshB-R, 773-
YaiT-F and yaiT-SwaI-R expands the upstreams the 400bp downstreams homology arm yaiT-up and 400bp homology arm yaiT-dn respectively.
PCR reaction systems include：Each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM
DNTP, 1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR reaction conditions are：94℃2min；98
DEG C 10s, 65 DEG C of 30s, 68 DEG C of 30s, hereafter each round cycle annealing temperature reduce by 1 DEG C, and 20 cycles, last 45 DEG C 10 are followed
Ring；68℃10min.Running gel recycles 400bp yaiT-up segments and 400bp yaiT-dn segments.
(3) using pTrc99a-gshB-ack-773 as template, yaiT-gshB-F and yaiT-773-R are primer, with KOD enzymes
Amplification includes the long segment ptrc-gshb-ack-773 of promoter, integrator gene and the resistant gene with the sites FRT.
yaiT-gshB-F：gtactgctaatcagcttgatgtttgacagcttatcatcga(SEQ ID NO:20)
773-yaiT-R：acgtaatcgctgctatggatgtttgtagaaacgcaaaaag(SEQ ID NO:21)
PCR reaction systems include：Each 0.3 μM of primer, template 50ng, 1 × KOD neo plus buffer, 0.2mM
DNTP, 1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR reaction conditions are：94 DEG C, 2min；
98 DEG C of 10s, 68 DEG C of 2min repeat 30 cycles；68℃10min.Running gel recycles 4kb target fragments.
(4) OVERLAP-PCR splices three segments, obtains integration box
Tri- fragment concentrations of electrophoresis detection yaiT-up, yaiT-dn, ptrc-gshB-ack-773, ensure three segments etc. as possible
Molar ratio.
PCR reaction systems include：1 × KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO4, 2%DMSO,
Each 0.3 μM of yaiT-up, yaiT-dn and ptrc-gshB-ack, yabp-SwaI-F and each 0.3 μM of yabp-SwaI-R, KOD neo
Plus1U, moisturizing to total system are 50 μ L, totally 6 pipe.
PCR reaction conditions are：94℃2min；98 DEG C of 10s, 65 DEG C of 30s, 68 DEG C of 2min30s, hereafter each round cycle annealing
Temperature reduces by 1 DEG C, and 20 cycles, last 45 DEG C 10 recycle；68℃10min.
Running gel recycles 5kb or so large fragment, is ptrc-gshB-ack-773 integration box, is concentrated into 10 μ L or so.
(5) Red homologous recombinations integrate Rosetta (DE3)
There is Rosetta (DE3) strain of pKD46 to be inoculated with conversion, 30 DEG C of overnight incubations.Next day is with 1:50 are inoculated in 50mL
SOB culture mediums (contain 100 μ g/mL Amp), while 20mM L-arabinoses are added, and 30 DEG C of cultures make pKD46 to OD600=0.6
Tri- albumen of upper Exo, Bet and Gam give full expression to.10min is pre-chilled on ice, bacterium solution is abandoned in 4 DEG C, 4000rpm centrifugations 10min
Clearly, with 10% glycerine centrifuge washing of precooling 3 times, it is condensed into the competent cell of 100-200 μ L.
Take 2-3uL integration box segments that competent cell is added, mixing is transferred in 0.2cm electric shock cups, is shocked by electricity with Bio-rad
Instrument does electrotransformation, and voltage 2.5kV, shock by electricity time 4-5s.It is rapidly added 1mL LB, 37 DEG C, 200rpm recovery 1h after electric shock, is coated with
Apr+Cm tablets, 37 DEG C of overnight incubations.
Bacterium colony PCR is with 4 pairs of primers identify the apramycin resistance clone grown respectively：
yaiTupup100：AGGGCGTTGGATTAAGTCTGTC(SEQ ID NO:22)
gshBdn：atcacgatgccgagcttgat(SEQ ID NO:23)
Correct integron can expand 1.2kb segments；
pTrcHisrev-F：agcctgatacagattaaatc(SEQ ID NO:24)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:25)
Correct integron can expand 1.2kb segments；
Apr-F：cgccattcgtattgcacg(SEQ ID NO:26)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:27)
Correct integron can expand 1.7kb segments；
Control Rosetta (DE3) cannot all expand band.
In the integron of screening, bright band can be expanded with pair of primers, size is correct；It can to primer with second and third
Very light band is expanded, but size is correct；Control does not all expand band.Obtain Escherichia coli transformation host Rosetta
(DE3)::gshB-ack。
(6) STH is at Rosetta (DE3)::Expression in gshB-ack
The recombinant plasmid pTrc99a-STH of aforementioned structure Calcium Chloride Methods are converted into above-mentioned Escherichia coli, host is transformed
Rosetta(DE3)::GshB-ack obtains Rosetta (DE3)::gshB-ack/pTrc99a-STH.
3, expression of the STH in the escherichia coli host Rosetta (DE3) for integrating ECack
By Acetokinase gene ECack (the NCBI accession number of Escherichia coli：CP001509.3) it is integrated into Rosetta
(DE3) sites yaiT of genome.
(1) structure of pTrc99a-ECack-773
Using genome of E.coli DNA as template, design primer expands 1.2kb ECack segments.
ECack-NcoI-F：CATGCCATGGgatcgagtaagttagtactg(SEQ ID NO:28)
ECack-BamHI-R：CGGGATCCtcaggcagtcaggcggct(SEQ ID NO:29)
PCR reaction systems include：Each 0.3 μM of primer, template 200ng, 1 × KOD neo plus buffer, 0.2mM
DNTP, 1.5mM MgSO4, KOD neo plus1U, benefit ddH2O to 50 μ L of total system.PCR reaction conditions are：94 DEG C, 2min；
98 DEG C of 10s, 68 DEG C of 45s repeat 30 cycles；68℃10min.
Running gel recycles 1.2kb target fragments, is purified after being handled with NcoI/BamHI (1 × Buffer Tango) double digestion
Recycling is connect overnight, chlorination with the pTrc99a-gshB-ack-773 segments of same digestion processing with T4DNA ligases in 16 DEG C
Calcium method converts DH5 α, is coated with Amp plate incubated overnights.It chooses monoclonal to be verified with NcoI/BamHI, is if 1.2kb segments can be cut out
Correctly, recombinant plasmid pTrc99a-ECack-773, plasmid map such as Figure 16 are obtained.
(2) side of the preparation and Red homologous recombinations integration Rosetta (DE3) of apramycin resistance integration box segment is carried
Method is the same as abovementioned steps 2 (2)-(5).
Bacterium colony PCR is with 2 pairs of primers identify the apramycin resistance clone grown respectively：
yaiTupup100：AGGGCGTTGGATTAAGTCTGTC(SEQ ID NO:30)
ECackdn：agtactaacttactcgatcc(SEQ ID NO:31)
Correct integron can expand 1.2kb segments；
Apr-F：cgccattcgtattgcacg(SEQ ID NO:32)
yaiTdndn：gagtagctcaagctaccatc(SEQ ID NO:33)
Correct integron can expand 1.7kb segments；
Control Rosetta (DE3) cannot all expand band.
In the integron of screening, bright band can be all expanded with these two pair primer, size is correct, is required.It obtains big
Host Rosetta (DE3) is transformed in enterobacteria::ECack.
(2) STH is at Rosetta (DE3)::Expression in ECack
The recombinant plasmid pTrc99a-STH of aforementioned structure Calcium Chloride Methods are converted into above-mentioned Escherichia coli, host is transformed
Rosetta(DE3)::ECack obtains strain Rosetta (DE3)::ECack/pTrc99a-STH.
(3) by pET24a-STH-opt conversion Rosetta (DE3)::ECack obtains Rosetta (DE3)::ECack/
pET24a-STH-opt；Convert Rosetta (DE3)::GshB-ack obtains Rosetta (DE3)::gshB-ack/pET24a-
STH-opt。
4, bacterial strain conversion capability
Strain 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 of gshB-ack/pET24a-STH-opt is tested.
Fermentation and bacterial cell disruption are the same as embodiment 1-12.
Conversion condition：React final volume 50ml (phosphate buffer 0.1mM, pH7.2)：L-cysteine：80mM, sweet ammonia
Acid：120mM, L-sodium：120mM, seven aqueous magnesium chlorides：40mM, acetyl phosphate dilithium salt：120mM, ATP：1mM, thalline are broken
Broken liquid：20%V/V.Phosphate buffer first is added in chemical reagent successively by above-mentioned formula, then adjusts pH7.2, bacterial cell disruption is added
Liquid, is packed into 250ml shaking flasks, is sealed after logical nitrogen, 30 DEG C, and 150rpm reactions take 500 μ l samples to be added in EP pipes, add 500
μ l10% hydrochloric acid stopped reactions.As a result such as the 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 |
It can be seen that Rosetta (DE3)/pTrc99a-STH/pMWK-gshB-ack has highest transformation efficiency.Its
The bacterial strain that it recombinantly expresses STH and ack also has relatively high transformation efficiency.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (11)
1. a kind of method producing glutathione, which is characterized in that the method includes：
(1) it is overexpressed glutathione bifunctional enzyme and acetokinase in Bacillus coli cells, obtains recombinant expression cell；Institute
The Escherichia coli stated are Rosetta (DE3), using expression plasmid pTrc99a as recombinant expression plasmid；With
(2) it is crushed the cell of (1), by 10~30%V/V of clasmatosis product and 80 ± 40mM of L-cysteine, glycine 120
120 ± 40mM of ± 40mM, Pidolidone or its salt, inorganic salts and ATP 1 ± 0.5mM hybrid reactions produce glutathione.
2. the method as described in claim 1, which is characterized in that in step (1), paddy Guang is overexpressed also in Bacillus coli cells
Sweet peptide synthetase.
3. method as claimed in claim 1 or 2, which is characterized in that in step (1), the glutathione bifunctional enzyme is compiled
Code gene and acetate kinase-encoding gene and/or glutathione synthetase-coding gene gshB are expressed in same Bacillus coli cells
In culture systems；Or it is expressed in different Escherichia coli culture systems.
4. method as claimed in claim 1 or 2, which is characterized in that step (1) includes：
(a) a kind of recombinant Bacillus coli cells are provided, the recombinant expression of following gene is included in the recombinant Bacillus coli cells
Box：Glutathione bifunctional enzyme encoding gene and acetate kinase-encoding gene, and selectivity are compiled containing glutathione synthetase
Code gene gshB；Or
Recombinant Bacillus coli cells are provided, including：A kind of recombinant expression cassettes comprising glutathione bifunctional enzyme encoding gene
The Bacillus coli cells and/or one kind of Bacillus coli cells and a kind of recombinant expression cassettes comprising acetate kinase-encoding gene include
The Bacillus coli cells of the recombinant expression cassettes of glutathione synthetase-coding gene gshB；With
(b) recombinant Bacillus coli cells for cultivating (a), to be overexpressed glutathione bifunctional enzyme and acetokinase.
5. the method as described in claim 1, which is characterized in that the glutathione bifunctional enzyme is derived from thermophilus
The glutathione bifunctional enzyme of bacterium (Streptococcus thermophilus).
6. the method as described in claim 1, which is characterized in that the acetokinase is derived from Lactobacillus sanfrancisco
(Lactobacillus sanfranciscensis) or the acetokinase of Escherichia coli (Escherichia coli).
7. the method as described in claim 1, which is characterized in that in step (2), the inorganic salts include：Magnesium salts, acetyl phosphorus
Hydrochlorate.
8. the method as described in claim 1, which is characterized in that in step (2), inorganic salts are in reaction system：Seven water chlorinations
Magnesium：40±30mM；Acetyl phosphate dilithium salt：120±40mM.
9. a kind of for producing the kit of glutathione, which is characterized in that the kit includes：
Recombinant expression carrier, the recombinant expression carrier include the recombinant expression cassettes of following gene：Glutathione bifunctional enzyme
Encoding gene and acetate kinase-encoding gene；Or including：The Bacillus coli cells of recombination containing the recombinant expression carrier；
The Escherichia coli are Rosetta (DE3), expression vector pTrc99a；With
L-cysteine, glycine, Pidolidone or its salt, inorganic salts and ATP；And theirs is final concentration of：L-cysteine
80 ± 40mM, 120 ± 40mM of glycine, Pidolidone or its salt 120 ± 40mM, 1 ± 0.5mM of inorganic salts and ATP.
10. kit as claimed in claim 9, which is characterized in that the recombinant expression carrier further includes following gene
Recombinant expression cassettes：Glutathione synthetase-coding gene gshB.
11. the kit as described in claim 9 or 10, which is characterized in that the inorganic salts include：Seven aqueous magnesium chlorides, second
Acyl phosphoric acid dilithium salt.
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| CN106834394B (en) * | 2017-01-20 | 2020-11-06 | 天津科技大学 | Preparation method of glutamine dipeptide |
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| CN111979206B (en) * | 2019-05-24 | 2021-08-17 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
| CN110373370B (en) * | 2019-07-16 | 2022-11-18 | 大连医诺生物股份有限公司 | Catalytic system coupled with ATP regeneration system and application of catalytic system in glutathione production process |
| CN110387379B (en) * | 2019-07-16 | 2022-11-22 | 大连医诺生物股份有限公司 | Mixed culture process and application of recombinant escherichia coli for producing glutathione |
| CN113005137B (en) * | 2021-02-25 | 2022-10-11 | 石河子大学 | Construction method of regulatory element with dual functions of starting and stopping, dual-function element library and application |
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