CN104059936B

CN104059936B - Preparation method of genetically engineered bacterium for synthesizing glutathione and product thereof

Info

Publication number: CN104059936B
Application number: CN201410183018.XA
Authority: CN
Inventors: 王伟; 唐星; 唐亮; 王玮玮; 杨燕; 周文龙; 朱义波
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-04-30
Filing date: 2014-04-30
Publication date: 2017-01-25
Anticipated expiration: 2034-04-30
Also published as: CN104059936A

Abstract

The invention discloses a preparation method of a genetically engineered bacterium for synthesizing glutathione. The preparation method comprises the following steps: (i) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase fusion protein into a host bacterium cell, and highly expressing the gene; (ii) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase into the host bacterium cell, and highly expressing the gene; (iii) knocking off the gamma-glutamyltranspeptidase of the host bacterium cell; (iv) knocking off an inositol-3-phosphatep synthase gene of the host bacterium cell; and completing the steps (i), (ii), (iii) and (iv) in any sequence, thereby obtaining the genetically engineered bacterium for synthesizing glutathione. The invention further relates to the prepared genetically engineered bacterium. The genetically engineered bacterium for synthesizing glutathione, which is disclosed by the invention, is not only high in efficiency in synthesizing glutathione, but also can greatly improve the conversion efficiency of exogenous cysteine, and can be applied to glutathione production.

Description

A kind of preparation method of genetic engineering bacterium for synthesizing glutathion and products thereof

Technical field

The present invention relates to bioengineering field, the system of more particularly to a kind of genetic engineering bacterium for synthesizing glutathion Preparation Method and products thereof.

Background technology

Glutathione (glutathione, gsh), i.e. γ-l- glutamy-l- cysteinyl-glycine, is a kind of by l- Glutamic acid, l- cysteine and glycine composition, it is widely present in biological internal biologically active tripeptide compound (meister a,anderson me.glutathione.annu rev biochem,1983,52:711–760).In all lifes In thing, glutathione with two kinds of existence forms, i.e. reduced glutathione (gsh) and oxidized form of glutathione (gssg), wherein The reduced glutathione playing a major role in body occupies the majority (akerboom tp, bilizer mm, sies h.the relationship of biliary gssg efflux and intracellular gssg content in perfused rat liver.j biol chem,1982,257:4248–4252).Glutathione have in vivo anti-oxidant, Remove function (carmel ho, the storz g.roles of the glutathione and such as free radical, suppression hiv thioredoxin-dependent reduction systems in the escherichia coli and saccharomyces cerevisiae responses to oxidative stress.annu rev microbiol, 2000,54:439–461；jahoor f,jackson a,gazzard b,et al.erythrocyte glutathione deficiency in symptom-free hiv infection is associated with decreased synthesis rate.j am j physio,1999,276(1):e205-e211).At present, it has been widely used in doctor The fields such as medicine, cosmetics, food processing.

As important bioactivator, current document report mainly has 3 biological conjunctions to glutathione in vivo One-tenth approach:

I. standard way

In most of eukaryotics and gramnegative bacterium, the biosynthesis of glutathione is mainly passed through two steps and is relied on Enzymic catalytic reaction (meister a.glutathione metabolism and its selective in atp Modification.j biol chem, 1988,263:17205 17208):

(1) l- glutamic acid and l- cysteine are in gamma glutamyl cysteine synthetase (gamma- Glutamylcysteine synthetase, γ-gcs, or gsh i) in the presence of synthesize gamma-glutamyl cysteine；

(2) gamma-glutamyl cysteine is in glutathione synthetase (glutathione synthetase, gs, or gsh Ii, in the presence of), generate glutathione with glycine reactant.Two-step reaction is as follows:

L-glutamate+l-cysteine+atp → l- γ-glutamyl-l-cysteine+adp+pi (reaction 1)

L- γ-glutamyl-l-cysteine+glycine+atp → gsh+adp+pi (reaction 2)

In the building-up process of glutathione, γ-gcs is subject to the feedback inhibition of end-product gsh, to avoid building up, And rate-limiting step (murata k, the kimura a.overproduction of being formed as this approach of γ-gc glutathione and its derivatives by genetically engineered microbial cells.biotechnol adv,1990,8:59–96).

Ii. bifunctional enzyme approach

In some pathogenic gram-positive bacteriums, such as Streptococcusagalactiae (streptococcus Agalactiae), Listeria (listeria monocytogenes), pasteurella (pasteurella Multocida) exist a kind of glutathione synthetase gamma glutamyl cysteine synthetase-glutathione synthetase (γ- Gcs-gs, gshf), this enzyme is a bifunctional enzyme, has gamma glutamyl cysteine synthetase and glutathione synthesis simultaneously The catalysis activity of enzyme, can individually be catalyzed generation (gopal s, the borovok i, ofer a, et al.a of glutathione multidomain fusion protein in listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.j bacteriol,2005,187:3839– 3847；janowiak be,griffith ow.glutathione synthesis in streptococcus agalactiae.one protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities.j biol chem,2005,280:11829–11839；vergauwen b,de vos d,van beeumen jj.characterization of the bifunctional gamma- glutamate-cysteine ligase/glutathione synthetase(gshf)of pasteurella multocida.j biol chem,2006,281:4380–4394).The size of γ-gcs-gs functional protein and γ-gcs and gs The zymoprotein size sum of this two enzymes is similar to.Therefore, certain evolutionary relationship is existed on biological evolution；But this enzyme pair The feedback insensitive of gsh, the biosynthetic course of reaction of gsh is also similar to that above-mentioned 2 independent functional protein enzymatics Course of reaction.

Iii. alternative route

The functional gene of gamma glutamyl cysteine synthetase (γ-gcs) is not contained in most of aerobic bacterias, but It remains to complete the biosynthesis of glutathione, meets the needs of vital movement.Therefore, other one must be contained in vivo at it Glutathione synthesis approach.Have document report recently, participate in Proline synthesis first functional protein enzyme prob (γ- Glutamic acid -5- phosphokinase), this enzyme coding functional protein can with glutamic acid for substrate formed gamma-glutamyl pyrophosphoric acid, γ - Glutamy pyrophosphoric acid can occur necleophilic reaction synthesis gamma-glutamyl cysteine with cysteine, can complete to be urged by γ-gcs The functional response being combined to, then catalyze and synthesize glutathione (veeravalli k, boyd d, iverson bl, et through gs al.laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.nature chemical biology,2010,7:101-105).Though this approach can synthesize Gsh, but reaction mechanism is different, the referred to as biosynthetic alternative route of glutathione.

The main production process of glutathione has chemical synthesis, immobilized cell (or enzyme) method and fermentation method.Chemistry closes It is ripe that one-tenth method produces glutathione technique, but it is many to there are high cost, reactions steps, needs chemical resolution and to there is environment dirty The problems such as dye；Production by Enzymes glutathione needs to obtain related catalysis enzyme system, needs to consume atp, leads to high cost；State at present The bacterial classification of inside and outside method for synthesizing glutathione by fermentation is mainly saccharomyces cerevisiae, candida utili etc., and produces bacterial strain and rely primarily on Traditional method for mutation breeding obtains.

The research report building high-yield glutathione bacterial strain using gene engineering method is more, mainly by glutathione mark The gamma glutamyl cysteine synthetase (gsh i) of quasi- biosynthesis pathway and glutathione synthetase (gsh ii) or double work( Can enzyme gshf gene overexpression in host cell, the activity of raising engineering cell Glutathione peptide synthetase and then raising paddy The synthesis level of the sweet peptide of Guang, the glutathione synthetase of this 2 approach respectively pichia pastoris phaff, saccharomyces cerevisiae and Succeed in Escherichia coli expression:

Escherichia coli are the genetic engineering modified of host: the colibacillary gamma-glutamyl of overexpression in Escherichia coli Cysteine synthase (also referred to as gsh a) and glutathione synthetase (also referred to as gsh b), recombinant bacterium jm109 (pvb303) The level of glutathione reaches 34.8mg/g wet thallus (liao xy, shen w, chen j, et al.improved glutathione production by gene expression in escherichia coli.lett appl microbiol,2006,43(2):211-214.)；Li Huazhong et al. also constructs gsh a and the plasmid conversion of gsh b gene is big Enterobacteria, makes glutathione be secreted into by fermentation process extracellular, fermentation yield is up to 7g/l.Although constructed engineering bacteria The level of glutathion production by fermentation is greatly improved, but sweat adds the toxic compounds such as toluene, is unfavorable for industry Metaplasia product (Li Huazhong, woods Jin Ping etc. there is structure and the synthetic reaction mistake of homoglutathion synthesizing activity recombination bacillus coli Journey. microorganism journal, 2001,41:16-23).Chinese Patent Application No. 201110003488.x discloses streptococcus thermophilus The recombinant Bacillus coli cells bl21/ of the difunctional glutathione synthetase gshf of (streptococcus thermophilus) Pet28a-gshf and jm109ptrc99a-gshf carries out abduction delivering, and three kinds of amino acid adopt disposable addition, repeatedly in batches Add or slow stream adds, the yield cultivating GSH-PX activity in different carbon sources is all greatly improved compared with the control, for example With glucose for the glutathione yield of three kinds of amino acid singles additions of carbon source as 3.4g/l, and the glutathione of batch fermentation produces Measure as 4.5g/l.

Pichia pastoris phaff is the genetic engineering modified of host: Chinese Patent Application No. 200810039695.9 He 201110343872.4 individually disclose colibacillary gsh a and gsh b is opened using composing type in pichia pastoris phaff Mover gap improves the expression of this 2 zymoproteins, improves the level of engineered strain synthesizing glutathion compared with control strain； Same Chinese Patent Application No. is 200710036487.9,200710192345.1,200810039695.9 and The 200910176777.2 gsh i and gsh ii also individually disclosing saccharomyces cerevisiae utilize composing type in pichia pastoris phaff Promoter gap improves the expression of this 2 zymoproteins, and engineered strain synthesis paddy Guang has also been significantly increased compared with control strain The level of sweet peptide；But synthesizing glutathion in the case of adding external source cysteine, although certain yield can also be reached, But still there is a problem of that transforms cysteine rate is low.Its main cause be in cell metabolism pond glutamic acid, cysteine, There is the process of osmosis of chemical small molecule it is difficult to realize gsh i and gsh in glycine and its intermediate gamma-glutamyl cysteine Ii2 walks rapidly efficiently being coupled of catalytic reaction, leads to catalytic efficiency relatively low.And there is the glutathione pair of 2 catalysis activities 2 two functional domain γ-gcs and gs of function synzyme gshf are sufficiently close on space structure, and two-step catalysis are connected more Closely, catalytic efficiency should be higher in theory, and middle precursor should be less；Enable gsh i and gsh ii2 and walk catalytic reaction It is coupled, have to overcome and permeate the catalysis leading between cell metabolism pond internal cause glycine and gamma-glutamyl cysteine equimolecular occasionally The low potentiality of connection efficiency；But Chinese Patent Application No. 201110369992.1 discloses Listeria monocytogenes (listeria Monocytogenes expression in pichia pastoris phaff for the bifunctional enzyme gshf gene), recombinant bacterium paddy under the conditions of shaking flask Guang sweet peptide yield is 4.13 times of wild type Pichi strain, and gsh yield is 190mg/l, and comparison gs115 wild mushroom gsh produces Amount is only 46mg/l (ge sl, zhu tc, li y.expression of bacterial gshf in pichia pastoris for glutathione production.appl environ microbiol,2012,78(15):5435- 5439).And the gsh i and gsh ii of saccharomyces cerevisiae go forward side by side in pichia pastoris phaff gs115 (pagpzhgsh) expression of restructuring Row high density fermentation, cell concentration reaches 98.15g (dry weight)/l, and the concentration of gsh reaches 4.15g/l (fei lw, yang y, chen sx.improved glutathione production by gene expression in pichia pastoris.bioprocess biosyst eng,2009,32:729-735).It can be seen that, bifunctional enzyme gshf finishes in Pasteur Gsh i and gshii that expression in red yeast is far below saccharomyces cerevisiae to the winding level of glutathione finishes red ferment in Pasteur Mother in expression when glutathione yield, this explanation bifunctional enzyme gshf catalysis activity relatively low.

Saccharomyces cerevisiae is that the genetic engineering modified of host: kiriyama etc. utilizes pgk promoter in saccharomyces cerevisiae yph499 The high expression gsh i and gsh ii of itself in cell, under conditions of Shaking culture, the yield of glutathione is 5.4mg/l/od, And compare as 1.83mg/l/od (kiriyama k, hara ky, kondo a.extracellular glutathione fermentation using engineered saccharomyces cerevisiae expressing a novel glutathione exporter.appl microbiol biotechnol,2012,96(4):1021-1027)；Fan etc. exists The only high expression gsh i of itself of saccharomyces cerevisiae ysf-31 cell, the yield of glutathione is 13.1mg/g stem cell, and to photograph Ratio improves 1.5 times of (fan x, he x, guo x, qu n, wang c, zhang b.increasing glutathione formation by functional expression of the gamma-glutamylcysteine synthetase gene in saccharomyces cerevisiae.biotechnol lett,2004,26(5):415-417).

Above-mentioned document report be primarily directed to height in different hosts cell for the glutathione standard biological route of synthesis Expression, thus improving the activity of glutathion inside cell synzyme, and then improves the level of cell synthesizing glutathion；But all the time Do not solve the difficult problem that gsh i and the reaction of gsh ii two-step catalysis are rapidly efficiently coupled, do not solve to add external source simultaneously yet The low problem of transforms cysteine efficiency.Moreover, this generation pyrogen in growth course of Escherichia coli, closes for fermentation Become glutathione and subsequent purification technique to bring difficulty, increased production cost.And pichia pastoris phaff is as protein expression System show its can high density fermentation and the advantage improving purpose product yield, but it has very oxybiontic preference by force, no Synthesis beneficial to reduced glutathione.

Content of the invention

In order to overcome the deficiencies in the prior art, it is an object of the invention to provide one kind has high-performance bio synthesis gluathione The genetic engineering bacterium of peptide, can dramatically improve the combined coefficient of glutathione, greatly improve raw material utilization efficiency, reduce Production cost.

For achieving the above object, the invention provides a kind of preparation side of the genetic engineering bacterium for synthesizing glutathion Method, comprising:

I) proceed to coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase Host Strains are intracellular The gene of fusion protein, and make its height expression；

Ii) encode gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein in intracellular the proceeding to of Host Strains Gene, and make its height expression；

Iii) knock out the gamma glutamyl transpeptidase gene of Host Strains cell；

Iv) knock out the inositol -3- phosphate synthase gene of Host Strains cell；

Described i), ii), iii), iv) obtain final product the described gene for synthesizing glutathion with after the completion of random order operation Engineering bacteria；

Described i) and ii) in connect peptide be made up of 6-34 amino acid residue, its formula is: g-x-g-y-g-z, wherein g For glycine, x, y, z respectively glycine, alanine, serine, 1～10 ammonia of threonine, glutamic acid and proline composition Base acid residue.

Preferably, described i), ii), iii), iv) in Host Strains be saccharomyces cerevisiae.

Preferably, described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein With the gene of gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein by constitutive promoter and/or induction type Promoter starts expression, and described constitutive promoter is any one in gap, pgk, adh1, tef, cyc1, tpi, described induction type Promoter is any one in gal1, gal2, gal3, gal5, gal7, gal10, cup1, met25.

Preferably, described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein With the gene of gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein, expression is started by constitutive promoter gap.

Preferably, coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein and γ- The gene of gamma-Glutamate kinase-connection peptide-glutathione synthetase fusion protein is by the high expression of integrated mode.

Preferably, described integrated mode height expression is real by using the integration site delta sequences of the high copy of yeast or λ dna Existing.

Preferably, described gamma glutamyl cysteine synthetase has the amino acid sequence shown in seq id no.2, its Preferably nucleotide sequence coded shown in seq id no.1.

Preferably, described glutathione synthetase has the amino acid sequence shown in seq id no.4 or seq id no.6 Row, wherein the amino acid sequence shown in seq id no.4 is preferably nucleotide sequence coded by seq id no.3；seq id Amino acid sequence shown in no.6 is preferably nucleotide sequence coded shown in seq id no.5.

Preferably, described gamma-glutamic acid kinases has the amino acid sequence shown in seq id no.8 or seq id no.10 Row, wherein the amino acid sequence shown in seq id no.8 is nucleotide sequence coded shown in seq id no.7；seq id Amino acid sequence shown in no.10 is preferably nucleotide sequence coded shown in seq id no.9.

Preferably, described connection peptide has the amino acid sequence shown in seq id no.12, and it is preferably by seq id Nucleotide sequence coded shown in no.11.

Preferably, described gamma glutamyl transpeptidase gene is knocked out by homologous recombination, wherein DNA homolog weight Group integrates frame using the homologous double-crossover with dna sequence shown in seq id no.13.

Preferably, described inositol -3- phosphate synthase gene is knocked out by homologous recombination, wherein DNA homolog weight Group integrates frame using the homologous double-crossover with dna sequence shown in seq id no.14.

Genetic engineering bacterium for synthesizing glutathion.

In the present invention, obtain two plants of genetic engineering bacteriums being used for synthesizing glutathion, Latin literary fame is saccharomyces Cerevisiae, deposit number is respectively cgmcc no.8932 and cgmcc no.8933, in preservation on the 18th in 03 month in 2014 In China Committee for Culture Collection of Microorganisms's common micro-organisms center (cgmcc), address is Chaoyang District, Beijing City North Star west Road 1 institute 3, Institute of Microorganism, Academia Sinica.

Compared to existing technology, the beneficial effects of the present invention is:

1. the present invention passes through the height of gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein The biosynthetic transcriptional control of host cell glutathione is broken in expression, makes intracellular glutathione synthetase increased activity, thus Improve the yield of glutathione；

2. the present invention is added by the high expression promotion of gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein Plus the conversion ratio of external source cysteine, and then improve the yield of glutathione；

3. the present invention passes through to knock out the catabolism that gamma glutamyl transpeptidase gene reduces intracellular glutathione, also simultaneously Knock out inositol -3- phosphate synthase gene, improve the structure of cell membrane, promote the exocytosis of glutathione；

4. use the method for the present invention to obtain genetic engineering bacterium, raw material utilization efficiency can be greatly improved, reduce production cost, It is applied to industrialized production.

Brief description

Fig. 1 is the identification of the pcr containing fusion gsh ii-gsh i positive colony；Wherein, m is dna molecular weight standard (trans2k plus ii dna marker), 1 is w303-1b, and 2 is w303-1b/g17^#, 3 is w303-1b/g20^#, 4 are W303-1b/g26#, 5 is w303-1b/g30^#；

Fig. 2 is gsh, γ-glu-cys, cys after hplc method analysis abd-f derivatization；

The fusion protein prob/t120i-gsh b that Fig. 3 purifies for sds-page analysis；Wherein, 1 is loading efflux, 2 For 20mm imidazoles elution samples, 3 is 50mm imidazoles elution samples, and 4 is 100mm imidazoles elution samples, and 5 is 300mm imidazoles wash-out Sample, m is Protein Marker；

Fig. 4 catalyzes and synthesizes the hplc analysis of gsh for fusion protein prob/t120i-gsh b；Wherein, 1 is gsh standard items, 2 gsh catalyzing and synthesizing for fusion protein prob/t120i-gsh b；

Fig. 5 is agarose gel electrophoresis analysis pcr amplification Escherichia coli gsh b_mThe dna fragment of mutant gene, wherein Swimming lane 1-13 is the pcr amplified production of different primers pair, and 1 is gshb1_7/gshb1_8, and 2 is gshb1_6/gshb1_9, and 3 are Gshb1_5/gshb1_10,4 is gshb1_4/gshb1_11, and 5 is gshb1_3/gshb1_12, and 6 is gshb1_2/gshb1_13, 7 is gshb2_6/gshb2_7, and 8 is gshb2_5/gshb2_8, and 9 is gshb2_4/gshb2_9, and 10 is gshb2_3/gshb2_ 10,11 is gshb2_2/gshb2_11, and 12 is gshb2_1/gshb2_12, and 13 is gshb1_1/gshb2_12, and m is dna molecule Amount standard (trans2k plus ii dna marker)；

Fig. 6 is to integrate fusion prob_m-gsh b_mThe pcr identification of engineering bacteria；Wherein, m is dna molecular weight standard (trans2k plus ii dna marker), 1 is negative control w303-1b/g；2-13 divides for positive colony w303-1b/gp Other 1^#,2^#,3^#,4^#,7^#,9^#,10^#,14^#,16^#,19^#,22^#,24^#；

Fig. 7 is the pcr identification of gamma glutamyl transpeptidase gene knockout engineered strain；Wherein, m is dna molecular weight standard (trans2k plus ii dna marker), 1 is negative control w303-1b/g, and 2 compare pgt2345- for positive plasmid Trp1,3 is w303-1b/g/gt^-24^#, 4 is w303-1b/gp/gt^-3^#, 5 is w303-1b/gp/gt^-4^#；

Fig. 8 is the pcr identification that inositol -3- phosphate synthase gene knocks out engineered strain；Wherein, m is dna molecular weight standard (trans2k plus ii dna marker)；1 is negative control w303-1b/g, and 2 is positive plasmid pino2345-ade2,3 For w303-1b/g/gt^-ino^-1^#, 4 is w303-1b/g/gt^-ino^-2^#, 5 is w303-1b/g/gt^-ino^-3^#, 6 is w303-1b/ g/gt^-ino^-4^#, 7 is w303-1b/gp/gt^-ino^-1^#, 8 is w303-1b/gp/gt^-ino^-2^#, 9 is w303-1b/gp/gt^-ino^- 3^#, 10 is w303-1b/gp/gt^-ino^-4^#；

Fig. 9 is to integrate fusion pro1-gsh b_mEngineering bacteria pcr identification；Wherein, m is dna molecular weight standard (trans2k plus ii dna marker), 1 is negative control w303-1b/g, and 2 is plasmid control tyr14-ug6-p_gap-pc- pro1-gsh b_m, 3 is positive colony w303-1b/gp1gt^-ino^-1^#；

Figure 10 utilizes ypd culture medium to add the impact to gsh yield for the amino acid in shake flask fermentation level for engineered strain.

Specific embodiment

The invention provides a kind of preparation method of the genetic engineering bacterium for synthesizing glutathion, comprising:

Iii) knock out the gamma glutamyl transpeptidase gene of Host Strains cell；

Iv) knock out the inositol -3- phosphate synthase gene of Host Strains cell；

The Host Strains adopting in the present invention can be Escherichia coli, (such as saccharomyces cerevisiae, Pasteur finishes red ferment to yeast cells Female) and the microbial cell, preferably saccharomyces cerevisiae such as filamentous fungi.

In the present invention, described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase merges egg The gene of white and gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein is by constitutive promoter and/or induction Type promoter starts expression, and described constitutive promoter is any one in gap, pgk, adh1, tef, cyc1, tpi, described induction Type promoter is any one in gal1, gal2, gal3, gal5, gal7, gal10, cup1, met25.

In the present invention, respectively using saccharomyces cerevisiae and pichia pastoris phaff constitutive promoter gap drive coding γ- GC-syn-connection peptide-glutathione synthetase fusion protein and gamma-glutamic acid kinases-connection peptide-paddy Guang The high expression jointly in host cell of the gene of sweet peptide synthetase fusion protein.

Described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein and γ-paddy The gene of histidine kinase-connection peptide-glutathione synthetase fusion protein is by the high expression of integrated mode.In the present invention, institute State coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein and gamma-glutamic acid kinases-company The gene integration connecing peptide-glutathione synthetase fusion protein in yeast delta sequences, wherein deposit in Yeast genome by delta sequences Copy at about 425, gene integration to this site enables the stable integration of its multicopy, and then reaches raising gene The purpose of expression.

Provide in the present invention and be separately encoded yeast shown in seq id no.3, seq id no.11, seq id no.1 Glutathione synthetase (gsh ii), connection peptide, the dna fragment clone of yeast gamma glutamyl cysteine synthetase (gsh i) Method, then obtains fusion gsh ii-gsh i, i.e. Homologous integration expression vector by way of gene is subcloned tyr14-hgyb/p_gap-sc/gsh2-gsh1/t_pgk, wherein using the composing type glyceraldehyde 3-phosphate dehydrogenase gene of saccharomyces cerevisiae Promoter gap starts its expression；Result is to have merged 7 histidine residue (seq id between expressed gsh ii and gsh i Amino acid sequence shown in no.12) so that 2 catalysis enzyme domains are spatially very close to thus effectively improve this 2 step The efficiency of cascade catalytic reaction, constructed engineering bacteria 2.3 times of glutathione output increased compared with the control.

Present invention also offers being separately encoded Escherichia coli gamma-glutamic acid kinase mutants (prob_m) (see seq id No.7), peptide, E. coli glutathione synzyme (gsh b are connected_m) (see seq id no.5) dna fragment cloning process, Then obtain fusion prob by way of gene is subcloned_m-gsh b_m, i.e. Homologous integration expression vector tyr14-ug6/ p_gap-pc/prob_m-gsh b_m/t_pgk, using pichia pastoris phaff composing type glyceraldehyde 3-phosphate dehydrogenase gene promoter gap Start its expression；Result is expressed prob_mWith gsh b_mBetween also merged 7 histidine residue (shown in seq id no.12 Amino acid sequence) so that 2 catalysis enzyme domains spatially very close to constructed engineering bacteria improves cysteine Transformation efficiency and glutathione yield.

Equally, obtain in the present invention encoding Saccharomyces cerevisiae gamma-glutamic acid kinases (pro1) (see seq id no.9) with E. coli glutathione synzyme (gsh b_m) fusion pro1-gsh b_m, i.e. Homologous integration expression vector tyr14- ug6/p_gap-pc/pro1-gsh b_m/t_pgk, also using the composing type glyceraldehyde 3-phosphate dehydrogenation of the high expression of pichia pastoris phaff Enzyme gene promoter gap starts its expression, and constructed engineering bacteria improves the transformation efficiency of cysteine and glutathione Yield.

The invention provides between gamma glutamyl cysteine synthetase and glutathione synthetase or gamma-glutamic acid Introduce, between kinases and glutathione synthetase, the pcr method connecting peptide (see seq id no.12), increase by 2 zymoprotein functions The flexible structure in domain, makes the two not because forming the change that fusion protein leads to space structure, reaches raising cascade catalytic reaction The purpose of efficiency.

The invention provides knocking out the technical scheme of gamma glutamyl transpeptidase gene by homologous recombination, used in it The homologous double-crossover of knockout gamma glutamyl transpeptidase gene integrate frame and there is the dna sequence of seq id no.13, containing 2 Length is respectively the gamma glutamyl transpeptidase genetic fragment of 1153bp and 431bp, and also containing a length is that 1163bp nutrition is mutual Mend selection markers trp1；Reduce the decomposition inducing because glutathion inside cell concentration is high by knocking out gamma glutamyl transpeptidase Metabolism, and then improve the yield of engineering bacteria glutathione.

Present invention also offers knock out the technical scheme of inositol -3- phosphate synthase gene by homologous recombination, wherein made The homologous double-crossover knocking out inositol -3- phosphate synthase gene integrates the dna sequence that frame has seq id no.14, contains 2 Individual length is respectively the inositol -3- phosphate synthase gene fragment of 866bp and 784bp, and also containing a length is 2516bp nutrition Complementation marks ade2；Reduce the combined coefficient of inositol by knocking out inositol -3- phosphate synthase, reach and change cell membrane knot Structure, promotes the efficiency to extracellular infiltration of glutathione, thus improving the synthesis level of glutathione.

In the present invention, two plants of genetic engineering bacteriums being used for synthesizing glutathion are obtained, deposit number is respectively cgmcc No.8932 and cgmcc no.8933, was preserved in China Committee for Culture Collection of Microorganisms on 03 18th, 2014 general Logical microorganism center (cgmcc), address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research Institute.

With reference to the accompanying drawings and examples the present invention is described in further detail.Those skilled in the art should manage Solution, these embodiments are only intended to illustration purpose, and do not limit the scope of the invention, in the bar without departing substantially from Claims scope Under part, those skilled in the art can carry out various modifications and improvements to various aspects of the present invention, these modification and improvement Fall within protection scope of the present invention.Unless specifically stated otherwise, various materials used in example below and reagent are all abilities Conventional material and reagent in domain, can be either commercially available by conventional, it is public that method therefor is those skilled in the art The conventional method known.

The extraction of embodiment 1 Yeast genome dna

Using ypd culture medium, (2%peptone, 1%yeast extract, 2%glucose, solid medium adds 1.5% agar) cultivate saccharomyces cerevisiae or pichia pastoris phaff, using the yeast of Beijing CoWin Bioscience Co., Ltd. Genome dna extracts kit (cat.no.cw0569) extracts its genome dna, and concrete operation step is as follows:

1. take 1-5ml yeast culture, 12000rpm is centrifuged 1 minute, collects thalline precipitates, and abandons supernatant.

2. the removal of yeast cell wall: add 600 μ l lyticase working buffer in thalline (using front inspection Look into addition beta -mercaptoethanol so as to final concentration of 0.1%), and add 5 μ l lyticase (10u/ μ l), fully mix.30℃ Process 30 minutes.4000rpm is centrifuged 10 minutes, abandons supernatant, collects precipitation.

3. add 200 μ l buffer gtl in precipitation, add 40mg bead (glass beads), be vortexed 5 minutes, 12000rpm is centrifuged 5 minutes, and supernatant is moved on in a new centrifuge tube.

4. add 20 μ l proteinase k, mix.55 DEG C of shaking baths, every 20-30 minute overturns and mixes during incubation Once；It is subsequently adding the rnase a solution that 4 μ l concentration are 100mg/ml, concussion mixes, and room temperature places 5-10 minute.

5.13000rpm is centrifuged 5 minutes, and careful supernatant of drawing is to new centrifuge tube.

6. add 200 μ l buffer gl, fully mix.70 DEG C are incubated 10 minutes, overturn therebetween and mix for several times.

7. add 200 μ l absolute ethyl alcohols, fully reverse mix, now it is possible that flocculent deposit.Of short duration centrifugation, makes pipe Liquid on wall and lid focuses on ttom of pipe.

8. step 7 resulting solution and precipitation are all added to the absorption having been charged into collecting pipe (collection tube) In post (spin column dm), if once solution can not be added, can proceed to several times.10000rpm is centrifuged 1 minute, outwells receipts Waste liquid in collector, adsorption column is placed back in collecting pipe.

9. add 500 μ l buffer gw1 in adsorption column, 10000rpm is centrifuged 1 minute, outwell useless in collecting pipe Liquid, adsorption column is placed back in collecting pipe.

10. add 500 μ l buffer gw2 (whether preoperation inspection has added absolute ethyl alcohol) in adsorption column, 10000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

11.12000rpm is centrifuged 2 minutes, outwells the waste liquid in collecting pipe.Adsorption column is placed in room temperature several minutes, with thorough Dry.

12. adsorption column is placed in a new centrifuge tube, vacantly adds 50-200 μ l to the middle part of adsorption column Buffer ge or aqua sterilisa, room temperature places 2-5 minute, and 10000rpm is centrifuged 1 minute, collects dna solution, -20 DEG C of preservation dna.

The structure of embodiment 2 gsh i-gsh ii Homologous integration expression vector

1. the clone of Homologous integration fragment δ dna fragment

Synthesize 4 primers according to the δ dna sequences Design of saccharomyces cerevisiae s288c (genbank number of registration is u20865) (to draw Thing nucleotide sequence is shown in seq id no.15-18):

ty_r1:5′-aatgcggtaccgcggccgcgagctgagaaatttgtgggt-3′

ty_r2:5′-tgtaatagggatatccacctaagtctcgagtctagaactagtggatcccc-3′

ty_r3:5′-acttaggtggatatccctattaca-3′

ty_r4:5′-aagctggagctcgcggccgcccgcggccgctataatgttg-3′；

With Yeast genome dna as template, carry out pcr expansion with 2 groups of primer ty_r1/ty_r2 and ty_r3/ty_r4 respectively Increase (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), grown Degree is respectively the dna fragment of 315bp and 199bp, purifies QIAquick Gel Extraction Kit (Beijing hundred Tyke biotechnology using multi-functional dna Co., Ltd) reclaim dna fragment from the gel containing purpose band after agarose gel electrophoresis, then respectively take 2 μ l (about 100ng) with pyrobest taq dna polymerase1u, 2 μ l reaction buffer, 1 μ l dntps (10mm), ddh₂The reagent such as o form 20 μ l reaction system, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 30s, 50 DEG C, 40s, 72 DEG C, 30s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).The dna fragment products after filling-in are recycled to be mould Plate, using primer ty_r1/ty_r4 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 35s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 490bp；Using restriction enzyme kpn i With sac i digestion and the cloned plasmids ez-t that is subcloned into identical inscribe ferment treatment (Kang Run sincere industry biotechnology in Beijing is limited Company) in, consistent with expected result through dna sequence verification, nucleotide sequence as shown in seq id no.19, that is, obtains centre Plasmid ez-tyr14；In Yeast genome, δ dna has 425 copies, and the genes of interest that can realize high copy by conversion is whole Close.

2. contain g418 and the cloning vector of hygromycin resistance selection markers builds

Method first with site-directed point mutation eliminates in carrier pug6 (genbank number of registration is af298793) upper table Reach restriction endonuclease sites pst i, the nru i in frame kanmx and hind iii, then utilize hind iii and ecor v limit Property restriction endonuclease processed is subcloned the dna fragment shown in seq id no.20 in plasmid ez-tyr14, that is, obtain carrier tyr14- ug6.

According to expression vector pan7-1 (genbank number of registration is z32698) upper encoding hygromycin phosphoric acid transferase gene 14 primer (primer cores of design synthesis based on the dna sequence of (hygromycin b phosphotransferase, hgyb) Nucleotide sequence is shown in seq id no.21-34), as shown in table 1.First with primer an7_1/an7_2, an7_3/an7_4, an7_ 5/an7_6, an7_7/an7_8, an7_9an7_10 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplify 289bp, 111bp, 117bp, 155bp, 204bp respectively Dna fragment, purifies QIAquick Gel Extraction Kit using multi-functional dna and reclaims dna fragment, then respectively take 2 μ l (about 100ng) and containing taq Kapa hifi hotstart readymix (2 ×) reagent of dna polymerase forms the reaction system of 20 μ l, carries out dna fragment Filling-in (95 DEG C, 2min；95 DEG C, 45s, 45 DEG C, 50s, 72 DEG C, 45s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in to be template, carry out pcr amplification (95 using primer an7_1/an7_10 DEG C, 5min；95 DEG C, 45s, 43 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtaining length is The dna fragment of 791bp；Reclaim dna fragment, using restriction enzyme nco i and spe i digestion and be subcloned the volume of 771bp Code frame to the clone pug6 with identical inscribe ferment treatment, that is, obtains plasmid pug6-an1；Utilize primer an7_11/an7_ simultaneously 12nd, an7_13/an7_14 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplify the dna fragment of 281bp, 354bp respectively, reclaim dna fragment, then respectively take 2 μ l (about 100ng) form the reactant of 20 μ l with kapa hifi hotstart readymix (2 ×) reagent of the polymerase of dna containing taq System, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 45s, 45 DEG C, 50s, 72 DEG C, 30s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in to be template, recycle primer an7_11/an7_14 Carry out pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 43 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 606bp；Purify and reclaim dna fragment, recycle restriction enzyme bgl ii and not I digestion is simultaneously subcloned the 576bp dna fragment containing part hgyb encoder block to the clone being processed with bamh i and not i digestion In pug6-an1, that is, obtain plasmid pug6-an2.Therefore, be mutated using the method for pcr restricted interior in hgyb encoder block Enzyme cutting site ecor i, pst i, nco i, nde i, bgl ii；Finally recycle hind iii and ecor v restriction enzyme Enzyme is subcloned the dna fragment shown in seq id no.35 in plasmid ez-tyr14, that is, obtain carrier tyr14-hgyb.

Table 1 synthesizes the pcr reaction primer that hygromycin phosphotransferase gene uses

3. the clone of saccharomyces cerevisiae pgk1 terminator

Triphosphoric acid glycerate kinase gene (phosphoglycerate kinase pgk) according to saccharomyces cerevisiae s288c (genbank number of registration is j01342) sequences Design synthesizes 4 primers (primer nucleotide sequences are shown in seq id no.36-39), As shown in table 2, with genome dna as template, carried out with primer pgk1_t1/pgk1_t4 the reaction of first round pcr (95 DEG C, 5min； 95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle first round pcr product For template, carried out with primer pgk1_t2/pgk1_t3 nido pcr reaction (nested-pcr) amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain a long dna being about 332bp Fragment.Purify purpose dna fragment from Ago-Gel, then use restriction enzyme xba i and hind iii digestion, more sub- 315bp dna fragment shown in clone seq id no.40 is to the carrier tyr14- being processed with identical digestion with restriction enzyme In ug6 and tyr14-hgyb, respectively obtain plasmid vector tyr14-ug6/tpgk and tyr14-hgyb/tpgk.

Table 2 expands the pcr reaction primer of terminator pgk

4. the clone of pichia pastoris phaff and saccharomyces cerevisiae gap promoter

According to pichia pastoris phaff glyceraldehyde 3-phosphate dehydrogenase (glyceraldehydes-3-phosphate Dehydrogenase) gene (genbank number of registration is fn392320) design 4 primers of synthesis (be shown in by primer nucleotide sequences Seq id no.41-44), as shown in table 3, with genome dna as template, carry out the first round with primer gap_pc1/gap_pc4 Pcr reaction (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min). Recycling first round pcr product be template, carried out with primer gap_pc2/gap_pc3 nested-pcr amplification (95 DEG C, 5min； 95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain a long about 886bp Dna fragment.Purify purpose dna fragment from Ago-Gel, then use restriction enzyme xba i and bamh i digestion, It is subcloned 874bp dna fragment as shown in seq id no.45 again to the carrier tyr14- with identical digestion with restriction enzyme ug6/t_pgkIn, obtain plasmid vector tyr14-ug6/t_pgk/p_gap-pc.

Using the primer gap_scp1/gap_scp2 (primer nucleotide sequences are shown in seq id no.46-47) of synthesis, such as table Shown in 3, and plasmid prs434gap (genbank number of registration be ab304854) for template carry out pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain a long about 715bp's Dna fragment.Purify purpose dna fragment from Ago-Gel, then use restriction enzyme xba i and bamh i digestion, then 703bp dna fragment shown in subclone seq id no.48 is to the carrier tyr14- with identical digestion with restriction enzyme hgyb/t_pgkIn, obtain plasmid vector tyr14-hgyb/p_gap-sc.

Table 3 expands the pcr reaction primer of pichia pastoris phaff and saccharomyces cerevisiae gap promoter

5. the clone of saccharomyces cerevisiae gsh ii gene

For convenience of the subclone of genetic fragment and the expression of fusion protein, according to saccharomyces cerevisiae s288c glutathione synthesis The sequences Design of enzyme (gsh) gene (genbank number of registration is nc_001147) synthesizes 16 primer (primer nucleotide sequences See seq id no.49-64), as shown in table 4, rite-directed mutagenesis is carried out by pcr reaction, eliminate restricted interior in dna sequence Enzyme cutting site hind iii, kpn i and spe i, whole gene fragment is divided into 3 fragments and is spliced.

The pcr amplification of gsh ii5 '-end dna fragment f1: with genome dna as template, entered with primer gsh2_1/gsh2_2 Row first round pcr reaction (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, carries out nested-pcr amplification (95 with primer gsh2_3/gsh2_4 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain gsh25 '- The length at end is the dna fragment of 665bp, is cloned on ez-t carrier by " ta " and carries out sequence verification, obtains plasmid ez- gsh2-f1.

The pcr amplification of dna fragment f2 in the middle of gsh ii: carry out continuously overlapping pcr using primer gsh2_5～gsh2_12 Amplification synthesis, initially with primer gsh2_8/gsh2_9 carry out dna fragment filling-in (95 DEG C, 4min；95 DEG C, 20s, 45 DEG C, 30s, 72 DEG C, 10s, 7cycles；72 DEG C, 10min；4 DEG C, 10min)；Then the dna fragment products after filling-in are utilized For template, using primer gsh2_7/gsh2_10 carry out the amplification of first round pcr (95 DEG C, 4min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 15s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Its amplified production organizes primer gsh2_6 and gsh2_11 as next Carry out the template of pcr amplified reaction, then carry out the second wheel pcr amplified reaction (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 20s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Primer gsh2_5 and gsh2_12 is subsequently recycled to carry out third round Pcr amplified reaction (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 25s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Amplification obtains dna fragment f2 of 288bp in the middle of gsh ii.

The amplification of gsh ii gene 3 '-end dna fragment f3: the pcr product being expanded with above-mentioned primer gsh2_1/gsh2_2 For template, using 2 groups of primers gsh2_13/gsh2_14, gsh2_15/gsh2_16 carry out nested-pcr amplification (95 DEG C, 5min；95 DEG C, 30s, 50 DEG C, 40s, 72 DEG C, 35s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), pcr amplification obtains 2 Length is respectively the dna fragment of 199bp and 391bp；Purify QIAquick Gel Extraction Kit using multi-functional dna and reclaim dna fragment, then respectively 2 μ l (about 100ng) and kapa hifi hotstart readymix (2 ×) reagent of the polymerase of dna containing taq is taken to form 20 μ l Reaction system, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 40s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in to be template, recycle primer Gsh2_13 and gsh2_16 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 40s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain dna fragment f3 that length is 590bp.

Gsh ii gene intermediate segment f2 and the splicing of 3 '-end dna fragment f3: purify reclaim reagent using multi-functional dna Box reclaims dna fragment, then takes intermediate segment f2 and the 3 '-end each 2 μ l of dna fragment f3 (about 50ng and 100ng respectively) and containing taq Kapa hifi hotstart readymix (2 ×) reagent of dna polymerase forms the reaction system of 20 μ l, carries out dna fragment Filling-in (95 DEG C, 2min；95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 45s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in to be template, recycle primer gsh2_5 and gsh2_16 to carry out pcr expansion Increase (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), grown Spend the dna fragment for 864bp；It is cloned on ez-t carrier by " ta " and carries out sequence verification, obtain plasmid ez-gsh2-f23. Restriction endonuclease ecor v is finally utilized to separate with spe i digestion ez-gsh2-f1 and using agarose gel electrophoresis and reclaim 5 '-hold Dna fragment f1, is then subcloned in the ez-gsh2-f23 through restriction endonuclease ecor v and nhe i process, finally gives seq id Gsh ii gene shown in no.3, that is, plasmid be named as ez-gsh ii, the amino acid sequence of this gene code is seq id no.4.The method that primer gsh2_16 is expanded by pcr is can be utilized to introduce at its 3 '-end during synthesis gsh ii gene Nucleotide sequence shown in seq id no.11, that is, the connection peptide amino acid sequence encoding be seq id no.12 and with gsh ii Amino acid sequence c- end is merged and is formed the flexible structure after fusion protein it is therefore an objective to increase with gsh i, keeps fusion protein can urge It is combined to the enzymatic activity of glutathione.

Table 4 expands the pcr reaction primer of glutathione synthetase (gsh ii) gene

6. the clone of saccharomyces cerevisiae gsh i gene

For convenience of the subclone of genetic fragment and the expression of fusion protein, according to saccharomyces cerevisiae s288c gamma-glutamyl half Guang The sequences Design of propylhomoserin synzyme (gsh1) gene (genbank number of registration is x85021) synthesizes 16 primer (prime nucleotide Sequence is shown in seq id no.65-80) as shown in table 5, rite-directed mutagenesis is carried out by pcr reaction, eliminate restricted in dna sequence Restriction enzyme site bamh i, ecor i, nco i, nde i and ssp i, whole gene fragment is divided into 3 fragments and is spliced.

The pcr amplification of gsh i5 '-end dna fragment f1: with genome dna as template, entered with primer gsh1_1/gsh1_16 Row first round pcr reaction (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, is entered with primer gsh1_2/gsh1_3 and gsh1_4/gsh1_5 respectively Row nested-pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment of length respectively 231bp, the 323bp at its 5 '-end, is purified back using multi-functional dna Receive kit and reclaim dna fragment, then respectively take the kapa hifi hotstart of 2 μ l (about 100ng) and the polymerase of dna containing taq Readymix (2 ×) reagent form 20 μ l reaction system, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 40s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in For template, recycle primer gsh1_2 and gsh1_5 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 40s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 536bp, ez- be cloned into by " ta " Carry out sequence verification on t carrier, obtain plasmid ez-gsh1-f1.

The pcr amplification of dna fragment f2 in the middle of gsh i: with the first round pcr product of primer gsh1_1/gsh1_16 as mould Plate, carried out with primer gsh1_6/gsh1_7 and gsh1_8/gsh1_9 respectively nested-pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 35s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the length difference in the middle of it Dna fragment for 283bp, 442bp, reclaims dna fragment using multi-functional dna purification kit, then respectively takes 2 μ l (about 100ng) form the reactant of 20 μ l with kapa hifi hotstart readymix (2 ×) reagent of the polymerase of dna containing taq System, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 45s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle the dna fragment products after filling-in to be template, entered using primer gsh1_6/gsh1_9 Row pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 45s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 705bp, be cloned on ez-t carrier by " ta " and carry out sequence verification, obtain plasmid ez-gsh1-f2.

The amplification of gsh i gene 3 '-end dna fragment f3: with the first round pcr product of primer gsh1_1/gsh1_16 as mould Plate, carries out nested-pcr expansion with primer gsh1_10/gsh1_11, gsh1_12/gsh1_13 and gsh1_14/gsh1_15 respectively Increase (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 35s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), expand Length to 3 '-end dna is respectively the dna fragment of 235bp, 137bp, 510bp, purifies QIAquick Gel Extraction Kit using multi-functional dna Reclaim dna fragment, then respectively take the kapa hifi of 2 μ l (respectively about 75ng, 50ng and 100ng) and the polymerase of dna containing taq Hotstart readymix (2 ×) reagent form 20 μ l reaction system, carry out dna fragment filling-in (95 DEG C, 2min； 95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 50s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).After recycling filling-in Dna fragment products be template, recycle primer gsh1_10/gsh1_15 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 50s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 853bp, pass through " ta " is cloned into and carries out sequence verification on ez-t carrier, obtains plasmid ez-gsh1-f3.

The splicing of gsh i full-length gene: using restriction enzyme ssp i and xba i digested plasmid ez-gsh1-f3, pure Change the dna fragment reclaiming its 3 '-end 847bp, be subcloned into the plasmid ez-gsh1-f2 being processed with sma i and xba i digestion, turn Change Escherichia coli screening positive clone, that is, obtain plasmid ez-gsh1-f23；Recycle restriction enzyme site ecor i on ez-t carrier Carry out digestion with the restriction enzyme site bgl ii in its 5 '-genetic fragment, purify the dna fragment reclaiming its 5 '-end 523bp, sub- It is cloned into the plasmid ez-gsh1-f23 being processed with bamh i and ecor i digestion, convert Escherichia coli screening positive clone, obtain final product To plasmid ez-gsh i；Finally give the gsh i gene shown in seq id no.1, the amino acid sequence of its coding is seq id no.2.

Table 5 expands the pcr reaction primer of gamma glutamyl cysteine synthetase (gsh i) gene

The structure of 7.gsh i-gsh ii Homologous integration expression vector

Using restriction enzyme nde i and bamh i digested plasmid ez-gsh ii, purify the dna piece reclaiming 1494bp Section, is subcloned into the plasmid vector tyr14-hgyb/p being processed with nde i and bamh i digestion_gap-sc, conversion Escherichia coli screening Positive colony, that is, obtain plasmid tyr14-hgyb/p_gap-sc/gsh2；Recycle restriction endonuclease bamh i and xba i digested plasmid ez- Gsh i, purifies the dna fragment reclaiming its 2053bp, is subcloned into the plasmid tyr14- being processed with bamh i and nhe i digestion hgyb/p_gap-sc/ gsh2, converts Escherichia coli screening positive clone, that is, obtain containing the gsh ii-gsh i fusion merging And the plasmid tyr14-hgyb/p of the Homologous integration for saccharomyces cerevisiae and expression_gap-sc/gsh ii-gsh i.

The conversion of embodiment 3 saccharomyces cerevisiae, the Screening and Identification of engineered strain and Methanogenesis

(1) preparation of competent yeast cells

1. it is stored in the w303-1b single bacterium colony on 4 DEG C of ypd flat board with oese picking, transfer in new ypd flat board, 30 DEG C of culture 2-3d, until grow single bacterium colony, activated spawn；

2. the w303-1b single bacterium colony of the new activation on picking ypd flat board is seeded to the 250ml equipped with 30ml ypd culture medium In triangular flask, 30 DEG C, 200rpm cultivates, to bacterium solution od₆₀₀For 1.0-1.2；

3. bacterium solution 4000rpm centrifugation 1min, abandons supernatant；

4. use the aseptic ddh of 30ml precooling₂O washing thalline, 4000rpm is centrifuged 1min, abandons supernatant, is repeated 3 times；

5. add the aseptic 1m sorbitol solution washing thalline of 30ml precooling, 4000rpm is centrifuged 1min, abandons supernatant, repeats 2 Secondary；

6. add the aseptic 1m sorbitol solution of 15ml precooling, mix, be sub-packed in ep pipe, often pipe 80 μ l, be placed in standby on ice With being also placed in -80 DEG C of preservations.

(2) the electricity conversion of yeast

1. 10～20 μ g expression vector tyr14-hgyb/p_gap-sc/ gsh ii-gsh i is linear through restriction endonuclease not i digestion After change, extracting precipitation, it is dissolved in the aseptic ddh of 20 μ l₂In o；

2. above-mentioned 20 μ l linearization plasmids are added in 80 μ l competent cell saccharomyces cerevisiae w303-1b, lightly mix Proceed in the electric revolving cup of precooling after conjunction, ice bath 5min；

3. electroporated；

4. it is rapidly added the 1m sorbierite of 1ml precooling on ice, after soft mixing, proceed in ep pipe, 30 DEG C of incubation 1h；

5. taking 300 μ l to coat on ypd flat board, being placed in 30 DEG C of culture 2-3d, until growing single bacterium colony.

(3) screening of positive strain

Single bacterium colony is grown on the ypd flat board after conversion, then single bacterium colony is transferred to the ypd that hygromycin b concentration is 4mg/ml On flat board, cultivate 2-3d；In order to detect whether gsh ii-gsh i fusion is integrated in saccharomyces cerevisiae w303-1b, then choose The bacterial strain growing is taken on resistant panel, the yeast being century Yeast genome dna extracts kit extraction screening transformant with health Genome dna, then with genome dna as template, using primer gap_scp1/pgk1_t3 carry out first round amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 4min；30cycles；72 DEG C, 10min；4 DEG C, 10min), with first round product For template, primer gsh2_5/gsh1_15 carry out the second wheel pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 3min；30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment of 2900bp, reclaim purpose fragment and be sequenced Identification obtains correct bacterial strain w303-1b/g, and (g represents p_gap-sc-gsh ii-gsh i-t_pgkExpression cassette) 17^#、20^#、26^#(see figure 1).

(4) acquisition of high-yield glutathione bacterial strain

1. select engineered strain, be seeded in the 250ml triangular flask containing 30ml ypd culture medium, 30 DEG C, 200rpm mistake Night cultivates；

2. above-mentioned bacterium solution 0.5ml is taken to be transferred in the 250ml triangular flask equipped with 50ml ypd culture medium, 30 DEG C, 200rpm Incubated overnight；

3. by step, bacterium solution 2. is transferred in the 500ml triangular flask equipped with 100ml ypd culture medium again, controls initial od₆₀₀=0.15, ten layers of gauze plug sealing, 30 DEG C, 200rpm cultivates.

4. sample every 24h, using w303-1b bacterial strain as comparison, quantitation is carried out to the glutathione of engineered strain synthesis Analysis, the final engineered strain obtaining high-yield glutathione.

The quantitative approach of embodiment 4 gsh

In experiment measurement yeast cells, the method for gsh content is hplc pre-column derivatization method:

1. the extraction of intracellular glutathione: ethanol extraction method.Take 1ml bacterium solution, 4000rpm is centrifuged 1min, abandons supernatant, thalline With distilled water cyclic washing 2 times.The ethanol of addition 1ml40% in clean thalline, 2h under the conditions of being placed in 30 DEG C, then 4000rpm is centrifuged 1min, takes 500 μ l supernatants to manage in new ep.Repeat 1 time.Finally obtain the thalline extract of 1ml.

2. the derivative reaction of gsh: take the thalline extract of 25 μ l and solution i (1% sulfosalicylic acid of 25 μ l (ssa), 1mm edta) it is placed in ep pipe, whirlpool mixes, and makes liquid concentrate on ttom of pipe through of short duration centrifugation；35 DEG C, lucifuge incubation 5 Minute；Solution ii [1mg/ml abd-f (4- (the sulfonamides) -7- fluoro- 2,1,3- benzo evil of 30 μ l is added in ep pipe Diazole) and 100 μ l borate buffer solution (0.1m borate, 1mm edta, ph9.3)], whirlpool mix, make through of short duration centrifugation Liquid concentrates on ttom of pipe；35 DEG C, lucifuge is incubated 10 minutes；Add the 2m hcl terminating reaction of 50 μ l.

3. reactant liquor, through 0.2 μm of membrane filtration, carries out hplc analysis.Analytical column be ymc-pack ods-a250 × 4.6mm.d.s-5 μm, 30nm.Fixing phase (a phase) is 0.1m acetate buffer, ph4.0；Mobile phase b is 100% acetonitrile；Analysis Condition is 0-15min, 8%b phase；16-20min, 8%-90%b phase, 21-25min90%b phase；26-30min, 90%-8%b Phase flow velocity 1ml min^-1, the method as shown in Figure 2 can using UV-detector well separate gsh, γ-gc, cys derivatization Product is it is ensured that the accuracy of gsh assay.Final acquisition contains the high engineering bacteria expressing fusion gsh ii-gsh i The yield that w303-1b/g cultivates 72h glutathione in shake flask fermentation level is 206.7mg/l, and control strain w303-1b paddy Guang sweet peptide yield is 89mg/l.

The expression of embodiment 5 Escherichia coli prob-gsh b fusion protein and activity identification

In order to detect the catalysis activity of gamma-glutamic acid kinases (prob)-glutathione synthetase (gsh b) fusion protein, Build Escherichia coli prob-gsh b fusion protein expression vector first, the abduction delivering carrying out fusion protein purifies, and sets up body Outer enzymic catalytic reaction system is it is determined that the mutant fusion protein that the thr120 of prob sports ile has and catalyzes and synthesizes The activity of glutathione, specific process is as follows:

1. genome of E.coli dna extracts: the bacterial genomes with reference to Tiangeng biochemistry (Beijing) Co., Ltd extract examination Agent box extracts the genome dna of Escherichia coli bl21 (de3), specifically comprises the following steps that

(1) take inoculum 1-5ml, 10000rpm centrifugation 1min (minute), remove supernatant.

(2) add 200 μ l buffer solution ga in bacterial sediment, vibrate and thoroughly suspend to thalline；Add 4 μ l rnase a (100mg/ml) solution, vibration 15sec (second), room temperature places 5min.

(3) add 20 μ l proteinase k solution in pipe, mix.

(4) add 220 μ l buffer solution gb, vibrate 15sec, 70 DEG C of placement 10min, solution becomes limpid, and brief centrifugation is to go The globule except cap wall.

(5) 220 μ l absolute ethyl alcohols are added, fully vibration mixes 15sec.

(6) previous step resulting solution and flocculent deposit are all added in an adsorption column cb3 (adsorption column puts into collecting pipe In), 12000rpm is centrifuged 30sec, outwells waste liquid, adsorption column cb3 is put in collecting pipe.

(7) 500 μ l buffer solution gd are added in adsorption column cb3,12000rpm is centrifuged 30sec, outwells waste liquid, by adsorption column Cb3 puts in collecting pipe.

(8) 600 μ l rinsing liquid pw are added in adsorption column cb3,12000rpm is centrifuged 30sec, outwells waste liquid, adsorption column Cb3 puts in collecting pipe.

(9) repeat step (8).

(10) adsorption column cb3 is put back in collecting pipe, 12000rpm is centrifuged 2min, outwells waste liquid.Adsorption column cb3 is placed in Room temperature places several minutes, thoroughly to dry remaining rinsing liquid in sorbing material.

(11) adsorption column cb3 is proceeded in a clean centrifuge tube, to the hanging dropping in the middle part 50 μ l of adsorbed film Elution buffer te, room temperature places 2-5min, and 12000rpm is centrifuged 2min, and solution is collected in centrifuge tube, that is, obtain gene Group dna, -20 DEG C save backup.

2. the clone of Escherichia coli prob gene

Sequence according to Escherichia coli bl21 (de3) gamma-glutamic acid kinase gene (genbank number of registration is am946981) Design 8 primers (primer nucleotide sequences are shown in seq id no.81-88) of synthesis, as shown in table 6.With genome dna as template, Carried out with primer prob_1/prob_4 first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, uses primer prob_2/ respectively Prob_3 carry out nested-pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment that length is 1144bp, reclaims dna using multi-functional dna purification kit Fragment, then utilizes restriction enzyme nde i and hind iii digestion dna fragment, is then subcloned into and uses identical restriction endonuclease In the carrier pet-28a processing, that is, obtain plasmid pet28-prob, sequence verification is consistent with expected result.For obtaining Thr120 sports the mutant of ile, with plasmid pet28-prob as template, using primer prob_5/prob_6 and prob_7/ Prob_8 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment that length is respectively 143bp, 279bp, purifies QIAquick Gel Extraction Kit using multi-functional dna Reclaim dna fragment, then respectively take the kapa hifi of 2 μ l (respectively may be about 50ng and 100ng) and the polymerase of dna containing taq Hotstart readymix (2 ×) reagent form 20 μ l reaction system, carry out dna fragment filling-in (95 DEG C, 2min； 95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 40s, 7cycles；72 DEG C, 10min；4 DEG C, 10min).After recycling filling-in Dna fragment products be template, using primer prob_5/prob_8 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 40s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment that length is 400bp, using restriction Property restriction endonuclease sal i and stu i digestion, corresponding dna fragment in displacement plasmid pet28-prob, obtain seq id no.89 institute The 120th threonine of the prob showing sports the mutant gene (t120i) of isoleucine, that is, obtain plasmid pet28-prob/ t120i.It is possible with the amino acid sequence c- end that primer prob_3 is encoded at it by pcr amplification in the building-up process of this gene Introduce the connection peptide amino acid sequence shown in seq id no.12.

Table 6 expands the pcr reaction primer of gamma-glutamic acid kinases (prob) gene

3. the structure of the clone of wild-type e. coli gsh b gene and fusion prob/t120i-gsh b

According to Escherichia coli bl21 (de3) glutathione synthetase (gsh b) gene, (genbank number of registration is np_ 417422) sequences Design synthesizes 4 primers (primer nucleotide sequences are shown in seq id no.90-93), as shown in table 7.With base Because group dna be template, carried out with primer gsh2_e1/gsh2_e4 the reaction of first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, respectively profit Carried out with primer gsh2_e2/gsh2_e3 nested-pcr amplification (95 DEG C, 5min；95 DEG C, 45s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment that length is 972bp, using multi-functional Dna purification kit reclaims dna fragment, using restriction enzyme bgl ii and xho i digestion dna fragment, is then subcloned To in the carrier pet28-prob/t120i being processed with restriction endonuclease bamh i and xho i digestion, obtain shown in seq id no.94 Encoding E. coli gsh b gene, obtain plasmid pet28-prob/t120i-gsh b, sequence verification and expected result one Cause.

Table 7 expands the pcr reaction primer of E. coli glutathione synzyme (gsh b) gene

4. the expression and purification of Escherichia coli prob/t120i-gsh b fusion protein

The abduction delivering of fusion protein:

Use cacl₂Conversion method, by plasmid pet28-prob/t120i-gsh b conversion Escherichia coli bl21 (de3), is containing There is overnight incubation on the lb solid medium flat board of kanamycins (50 μ g/ml), picking monoclonal is inoculated into containing kanamycins In the 30ml lb culture medium of (50 μ g/ml), 37 DEG C, 200rpm cultivates to od₆₀₀To 1.0 about；By 1% inoculum concentration switching Contain in kanamycins and the lb culture medium of ampicillin sodium to 100ml, in 37 DEG C, 200rpm continuation culture to od₆₀₀Arrive 0.6 about, add final concentration 1mm iptg derivant to express 18 hours in 16 DEG C of low temperature inductions, 8000rpm centrifugation 1min will receive Collection thalline, abandons culture medium.

The purifying of fused protein:

(1) sample treatment: add 25ml lysis buffer (0.2m pbs, 1mm in the 30ml centrifuge tube of collects thalline Pmsf, ph8.0), with clean glass bar, thalline is stirred evenly, be placed in ice, carry out ultrasonication, 12,000rpm4 DEG C of centrifugations 15 Minute, supernatant is transferred in new 1.5ml microcentrifugal tube, 16000rpm is centrifuged 20 minutes, supernatant is taken out through 0.2um's Membrane filtration, removes impurity, just can carry out affinitive layer purification；

(2) process of nickel affinity chromatography post: wash post, then splitting with 5-10 times of column volume with the distilled water of 5-10 times of column volume Solution buffer solution is balanced, and prepares loading；

(3) it is added to slowly adherent for protein sample in post, carefully avoids hanging nickel glue, affect joint efficiency, with The flow velocity of 0.5ml/min releases loading efflux；

(4) imidazole gradient wash-out: respectively with 5mm, the imidazole concentration of 10mm, 15mm, 20mm, 50mm, 100mm, 300mm takes off Wash each 100ml of chromatographic column, collect each eluent and carry out sds-page electrophoresis detection (see Fig. 3), result shows to purify the sample obtaining Product purity is higher, with being consistent of expected fusion protein molecule 76kda.

5. the catalysed in vitro activity analysis of Escherichia coli prob/t120i-gsh b fusion protein

Elution samples containing imidazoles bag filter dialysed overnight, elution buffer is 0.2m pbs (ph8.0), utilizes Bio-rad protein quantification kit (cat no.500-0006) carries out protein quantification, takes 100 μ l (about 500 μ g) and 100 μ l's Amino acid and atp mixture mix [the mixed system configuration of amino acid and atp: atp (60mm), glutamic acid (60mm), glycine (60mm), cysteine (40mm), pbs buffer solution (0.2m ph=8)], 37 DEG C are reacted 6 hours.After reaction terminates, will react Sample heating is denatured by boiling, and 12000rpm is centrifuged 2min, through 0.2 μm of membrane filtration, carries out hplc analysis (hitachi l- 2000).Grace vydac polypeptide analytic type c₁₈Post (250mm × 4.6mm, 5 μm, 30nm) carry out gradient elution；Ultraviolet is examined Survey: 210nm；Flow velocity: 1ml/min；Column temperature: 25 DEG C, solvent is fixing phase a (0.05%tfa, v/v), mobile phase b (acetonitrile Acn90%, 0.05%tfa (trifluoroacetic acid), v/v), chromatographic condition elutes 15min for 2%b phase.Through hplc analysis result as schemed Using the catalyst system and catalyzing that the fusion protein prob/t120i-gsh b purifying sets up, there is the activity catalyzing and synthesizing gsh shown in 4.

Embodiment 6 E. coli mutant type prob_m-gsh b_mThe structure of Homologous integration expression vector

Because Escherichia coli and genes of brewing yeast coding have Preference, difference is very big, for realizing E. coli fusion High expression in gene prob/t120i-gsh b saccharomyces cerevisiae, this fusion is sported full the volume of suitable Yeast expression Code frame.

1. the optimization of Escherichia coli gsh b gene yeast preference coding

According to Escherichia coli bl21 (de3) glutathione synthetase (gsh b) gene, (genbank number of registration is np_ 417422) sequence designs synthetic primer (primer nucleotide sequences are shown in seq id no.95-119) for source, as shown in table 8, leads to Synthesizing, concrete grammar is as follows, whole dna fragment is divided into former and later two parts and is respectively synthesized to cross continuously overlapping pcr method:

The 5 ' of gsh b mutator-end dna fragment: carry out dna fragment initially with primer gshb1_7/gshb1_8 Filling-in (95 DEG C, 4min；95 DEG C, 10s, 45 DEG C, 30s, 72 DEG C, 10s, 7cycles；72 DEG C, 10min；4 DEG C, 10min)； Then utilize the dna fragment products after filling-in to be template, carry out the expansion of first round pcr using primer gshb1_6/gshb1_9 Increase (95 DEG C, 4min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 15s, 30cycles；72 DEG C, 10min；4 DEG C ,+∞)；Its amplification is produced Thing carries out the template of pcr amplified reaction as next group primer gshb1_5/gshb1_10, then carries out the second wheel pcr amplified reaction (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 20s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Subsequently successively The primer carrying out pcr amplification is gshb1_4/gshb1_11, gshb1_3/gshb1_12, gshb1_2/gshb1_13；Often extend The pcr amplified reaction of one group of primer, the extension of time of its pcr reaction increases 5s, takes turns pcr amplified reaction through 5, and amplification obtains about The dna fragment of 508bp, is named as gshb1_213.

The 3 ' of gsh b mutator-end dna fragment: carry out dna fragment initially with primer gshb2_6/gshb2_7 Filling-in (95 DEG C, 4min；95 DEG C, 10s, 45 DEG C, 30s, 72 DEG C, 10s, 7cycles；72 DEG C, 10min；4 DEG C, 10min)； Then utilize the dna fragment products after filling-in to be template, carry out the expansion of first round pcr using primer gshb2_5/gshb2_8 Increase (95 DEG C, 4min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 15s, 30cycles；72 DEG C, 10min；4 DEG C ,+∞)；Its amplification is produced Thing carries out the template of pcr amplified reaction as next group primer gshb2_4/gshb2_9, then carries out the second wheel pcr amplified reaction (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 20s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Subsequently successively The primer carrying out pcr amplification is gshb2_3/gshb2_10, gshb2_2/gshb2_11, gshb2_1/gshb2_12；Often extend The pcr amplified reaction of one group of primer, the extension of time of its pcr reaction increases 5s, takes turns pcr amplified reaction through 5, and amplification obtains about The dna fragment of 484bp, is named as gshb2_112.

The splicing of full-length gene: purify QIAquick Gel Extraction Kit using multi-functional dna and reclaim gshb1_213 and gshb2_112dna Fragment, then respectively takes the kapa hifi hotstart readymix (2 of 2 μ l (about 100ng) and the polymerase of dna containing taq ×) reagent form 20 μ l reaction system, carry out dna fragment filling-in (95 DEG C, 2min；95 DEG C, 40s, 45 DEG C, 50s, 72 DEG C, 1min, 7cycles；72 DEG C, 10min；4 DEG C, 10min).The dna fragment products after filling-in are recycled to be template, Recycle primer gshb1_1/gshb2_12 carry out pcr amplification (95 DEG C, 5min；95 DEG C, 40s, 43 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), obtain the dna fragment (see Fig. 5) that length is 977bp, load be cloned into by ta Carry out sequence verification on body ez-t, obtain plasmid ez-gsh b_m；Result obtains the Escherichia coli paddy Guang shown in seq id no.5 Sweet peptide synthetase gene mutation body (gsh b_m), the amino acid sequence of its coding is seq id no.6.

Table 8 E. coli glutathione synthesizes the pcr reaction primer of (gsh b) yeast preference encoding gene

2. the optimization of Escherichia coli prob mutant gene yeast preference coding

According to Escherichia coli bl21 (de3) gamma-glutamic acid kinases (prob) gene (genbank number of registration is am946981) Sequence designs synthetic primer (primer nucleotide sequences are shown in seq id no.120-147) for source, as shown in table 9, by continuous Synthesizing, specific experiment method of operating is as follows, whole dna fragment is divided into two fragments and is respectively synthesized for overlapping pcr method:

The 5 ' of prob mutator-end dna fragment: carry out the benefit of dna fragment initially with primer prob1_5/prob1_6 Redress should (95 DEG C, 4min；95 DEG C, 10s, 45 DEG C, 30s, 72 DEG C, 10s, 7cycles；72 DEG C, 10min；4 DEG C, 10min)；So Utilize the dna fragment products after filling-in to be template afterwards, carry out first round pcr using primer prob1_4/prob1_7 Amplification (95 DEG C, 4min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 15s, 30cycles；72 DEG C, 10min；4 DEG C ,+∞)；Its amplification Product carries out the template of pcr amplified reaction as next group primer prob1_3/prob1_8, then it is anti-to carry out the second wheel pcr amplification Should (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 20s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；Subsequently according to The secondary primer carrying out pcr amplification is prob1_2/prob1_9, prob1_1/prob1_10；Often extend the pcr amplification of one group of primer Reaction, the extension of time of its pcr reaction increases 5s, takes turns pcr amplified reaction through 4, and amplification obtains the dna fragment of about 408bp；Pure Change and reclaim dna fragment, be then cloned on carrier ez-t by ta and carry out sequence verification, obtain plasmid ez-prob1.

The 3 ' of prob mutator-end dna fragment: carry out dna fragment initially with primer prob2_9/prob2_10 Filling-in (95 DEG C, 4min；95 DEG C, 10s, 45 DEG C, 30s, 72 DEG C, 10s, 7cycles；72 DEG C, 10min；4 DEG C, 10min)； Then utilize the dna fragment products after filling-in to be template, carry out the first round using primer prob2_8/prob2_11 Pcr amplification (95 DEG C, 4min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 15s, 30cycles；72 DEG C, 10min；4 DEG C ,+∞)；Its Amplified production carries out the template of pcr amplified reaction as next group primer prob2_7/prob2_12, then carries out the second wheel pcr expansion Increasing reaction (95 DEG C, 5min；95 DEG C, 30s, 45 DEG C, 40s, 72 DEG C, 20s, 30cycles；72 DEG C, 10min；4 DEG C, 10min)；With Carry out successively afterwards pcr amplification primer be prob2_6/prob2_13, prob2_5/prob2_14, prob2_4/prob2_15, prob2_3/prob2_16、prob2_2/prob2_17、prob2_1/prob2_18；The pcr amplification often extending one group of primer is anti- Should, the extension of time of its pcr reaction increases 5s, takes turns pcr amplified reaction through 8, and amplification obtains the dna fragment of about 735bp；Purify Reclaim dna fragment, be then cloned on carrier ez-t by ta and carry out sequence verification, obtain plasmid ez-prob2.Finally utilize On carrier, the restriction enzyme site hpa i at restriction enzyme site hind iii and dna fragment 3 '-end carries out digested plasmid ez-prob1, Purify and reclaim dna fragment, then carry out even with through the vector plasmid ez-prob2 that restriction endonuclease hind iii and stu i digestion are processed Connect, conversion Escherichia coli screening obtains positive colony ez-prob_m, sequence verification, that is, obtain being suitable for shown in seq id no.7 Prob in yeast cell to express_mMutant, the amino acid sequence of its coding is seq id no.8.The building-up process of this gene In be possible with primer prob2_18 and expand the amino acid sequence c- end encoding at it by pcr introducing shown in seq id no.12 Connection peptide amino acid sequence it is therefore an objective to increase with gsh b_mForm the flexible structure after fusion protein, keep fusion protein energy Catalyze and synthesize the enzymatic activity of glutathione.

The pcr reaction primer of table 9 gamma-glutamic acid kinases (prob) yeast preference encoding gene

3. mutated genes prob_m-gsh b_mYeast homologous integrating expression vector build and yeast conversion

Use restriction enzyme bgl ii and nhe i digested plasmid ez-gsh b first_m, purify and reclaim encoding E. coli gsh b_mThe dna fragment of reading frame 961bp, the carrier tyr14-ug6/t processing with identical digestion_pgk/p_gap-pcIn, obtain plasmid Carrier tyr14-ug6/t_pgk/p_gap-pc/gsh b_m.Then recycle restriction endonuclease nde i and bamh i digested plasmid ez-prob_m, Purify to reclaim and contain Escherichia coli prob_mReading frame and coding connect the dna fragment of nucleotides 1121bp of peptide with through nde i and The plasmid vector tyr14-ug6/t that bgl ii digestion is processed_pgk/p_gap-pc/gsh b_mIt is attached, Escherichia coli are screened for conversion To positive colony tyr14-ug6/t_pgk/p_gap-pc/prob_m-gsh b_m, that is, obtain the fusion being suitable for saccharomyces cerevisiae expression prob_m-gsh b_mMutant, has merged therebetween the connection peptide shown in seq id no.12, strengthens two different catalytically actives Functional domain prob_mWith gsh b_mFlexible structure, the two space structure can be made neighbouring simultaneously, improve cascade catalytic reaction effect Rate.

Yeast conversion: be integrated with the fusion gsh ii-gsh i engineered strain of yeast using the conversion of liac conversion method w303-1b/g17^#(g represents p_gap-sc-gsh ii-gsh i-t_pgkExpression cassette), first with restriction endonuclease not i plasmid tyr14- ug6/t_pgk/p_gap-pc/prob_m-gsh b_mLinearization for enzyme restriction, conversion process is as follows:

(1) choose monoclonal and connect bacterium in 25ml liquid ypd culture medium, 30 DEG C, 250rpm cultivates 16-18h to od₆₀₀It is about 0.8-1.0.

(2) 1500rpm centrifugation 5min harvesting (1.5ml ep pipe), 1500rpm centrifugation after 750 μ l aqua sterilisa cleanings 5min, abandons supernatant.

(3) every effective 30 μ l, 100mm liac are resuspended, and 12000rpm is centrifuged 15s, abandons supernatant, 50 μ l, 100mm liac weight Outstanding as competent cell.

(4) 1ml single-stranded salmon essence carrier dna (ss carrier dna) 2mg/ml boils 5min, is immediately placed in cold on ice But.

(5) competent cell 12000rpm centrifugation 15s, abandons liac..

(6) it is sequentially added into 240 μ l500g/l peg (peg3350)；36μl liac(1m)；25μl ssdna；50 μ l lines Property plasmid (10-20 μ g), whirlpool acutely mixes 1min.

(7) 30 DEG C of stationary incubation 30min, 42 DEG C of water-bath thermal shock 60min, reverse mixing is for several times.

(8) supernatant is abandoned in centrifugation, adds ypd culture medium rejuvenation 2h, is applied to the ypd screening flat board containing 100 μ g/ml g418, 30 DEG C of culture 3d.Then picking monoclonal carries out the positive colony that high copy is integrated in the ypd screening flat board of 4mg/ml g418 Secondary screening.

4. integrate mutated genes prob_m-gsh b_mEngineered strain screening and Methanogenesis

By the monoclonal on the ypd flat board being grown in high concentration g418, cultivated using ypd fluid nutrient medium, carried out Screening.Extract its genome dna using Yeast genome dna purification kit, then carry out pcr identification, first with primer Prob1_1/gshb2_10 carry out the reaction of first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 1min, 72 DEG C, 2min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, uses primer prob1_2/ respectively Gshb2_9 carry out nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 1min, 72 DEG C, 2min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), the dna fragment obtaining that length is 1956bp can be expanded in theory, agarose gel electrophoresis is analyzed Result such as Fig. 6, is consistent with expected result, and 12 being screened clone is to be integrated with fusion prob_m-gsh b_m's Positive colony, engineered strain is named as w303-1b/gp, and (g represents p_gap-sc-gsh ii-gsh i-t_pgkExpression cassette, p represents p_gap-pc-prob_m-gsh b_m-t_pgkExpression cassette).

Picking positive colony carries out ypd Liquid Culture, carries out the yield in shaking flask horizontal analysis glutathione first:

4. sample every 24h, quantitative analysis is carried out to the glutathione of engineered strain synthesis, test result indicate that in shaking flask Fermentation level fermented and cultured 72h contains high expression fusion gsh ii-gsh i and prob_m-gsh b_mEngineering bacteria w303- The yield of 1b/gp glutathione is 309.46mg/l, and prob is described_m-gsh b_mHigh expression still can promote the conjunction of glutathione Become.

The structure of embodiment 7 gamma glutamyl transpeptidase gene knockout Homologous integration frame

1. gamma glutamyl transpeptidase DNA homolog integrates the clone of fragment

Sequences Design synthesis according to saccharomyces cerevisiae gamma glutamyl transpeptidase gene (genbank number of registration is u17243) Primer (primer nucleotide sequences are shown in seq id no.148-153), as shown in table 10.With genome dna as template, use primer Gt_1/gt_6 carry out the reaction of first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles； 72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, is carried out with primer gt_2/gt_3 respectively Nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains gamma glutamyl transpeptidase gene 5 '-end dna piece that length is the 1181bp shown in seq id no.154 Section, reclaims dna fragment using multi-functional dna purification kit, then utilizes restriction enzyme kpn i and bamh i digestion Dna fragment, is then subcloned in the carrier ez-t with identical inscribe ferment treatment, sequence verification obtains, that is, obtain plasmid ez- gt23.

First round pcr product also with primer gt_1/gt_6 is template, carries out nested- using gt_4/gt_5 Pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 30s, 30cycles；72 DEG C, 10min；4 DEG C, 10min), It is the gamma glutamyl transpeptidase gene 3 '-end 478bp dna fragment shown in seq id no.155 that amplification obtains length, using many Function dna purification kit reclaims dna fragment, then utilizes restriction enzyme bamh i and sac i digestion dna fragment, so It is subcloned into afterwards in the carrier ez-gt23 with identical inscribe ferment treatment, sequence verification obtains, that is, obtain plasmid ez-gt2345.

The pcr reaction primer of table 10 gamma-glutamyl gene

2. the structure of the clone of selection markers trp1 and Homologous integration carrier

According to the genotype of host cell w303-1b, choose selection markers trp1 gene as gamma glutamyl transpeptidase base Because of the selection markers knocking out, design synthetic primer (primer nucleotide sequences are shown in seq id no.156-157), as shown in table 11, Carry out pcr amplification using primer trp1_1/trp1_2 and the plasmid containing this selection markers for pgbkt7 template and obtain such as seq 1195bp dna fragment shown in id no.158.Reclaim dna fragment using multi-functional dna purification kit, then use restriction endonuclease Bamh i and sal i digestion dna fragment, are then subcloned in the carrier ez-gt2345 with identical inscribe ferment treatment, are struck Homologous double-crossover except gamma glutamyl transpeptidase gene integrates frame, and sequence verification obtains the dna sequence as shown in seq id no.13 Array structure, that is, obtain plasmid ez-gt2345-trp1.

The pcr reaction primer of table 11 selection markers trp1 gene

Embodiment 8 gamma glutamyl transpeptidase gene knockout and Methanogenesis

(g represents to incorporate fusion gsh ii-gsh i engineered strain w303-1b/g using the conversion of liac conversion method p_gap-sc-gsh ii-gsh i-t_pgkExpression cassette) and incorporate fusion gsh ii-gsh i, prob_m-gsh b_mEngineered strain (g represents p to w303-1b/gp_gap-sc-gsh ii-gsh i-t_pgkExpression cassette, p represents p_gap-pc-prob_m-gsh b_m-t_pgkExpression Frame).

First with restriction endonuclease not i plasmid ez-gt2345-trp1 linearization for enzyme restriction, conversion process is as follows:

(4) 1ml ss carrier dna2mg/ml boils 5min, is immediately placed in cooled on ice.

(5) competent cell 12000rpm centrifugation 15s, abandons liac..

(7) 30 DEG C of stationary incubation 30min, 42 DEG C of water-bath thermal shock 60min, reverse mixing is for several times.Supernatant is abandoned in centrifugation, adds ypd Culture medium rejuvenation 2h.

(8) supernatant is removed in centrifugation, then uses sc trp^-Auxotroph culture medium rinses 2 times, then uses sc trp^-Auxotrophy Type culture medium is resuspended, is applied to sc trp^-In auxotroph solid medium screening flat board, 30 DEG C of culture 3d.Then picking list Clone's culture, purifies its genome dna using Yeast genome dna extracts kit, carries out knock-out bacterial strain using pcr method Identification.

(9) (primer nucleotide sequences are shown in seq id to utilize gamma glutamyl transpeptidase DNA homolog area design synthetic primer No.159-162), as shown in table 12, using primer gt_t1/gt_t4 carry out first round amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is mould Plate, carried out with primer gt_t2/gt_t3 respectively nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment that length is 1315bp, and control strain It is only capable of amplifying the dna fragment of 692bp, result two kinds of engineered strains as shown in Figure 7 all obtain gamma glutamyl transpeptidase gene Knock out w303-1b/g/gt^-And w303-1b/gp/gt^-.

Picking positive colony carries out ypd Liquid Culture, carries out the volume analysis in shaking flask horizontal analysis glutathione first:

4. sample every 24h, quantitative analysis is carried out to the glutathione of engineered strain synthesis, experiment shows in shake flask fermentation Horizontal fermented and cultured 72h knocks out gamma glutamyl transpeptidase genetic engineering bacterium w303-1b/g/gt^-And w303-1b/gp/gt^-Paddy Guang The yield of sweet peptide is respectively 486.7mg/l and 728.5mg/l, illustrates that inactivation gamma glutamyl transpeptidase gene can be greatly Promote the synthesis of glutathione.

The pcr identification reaction primer of table 12 gamma glutamyl transpeptidase gene knockout

The structure of embodiment 9 inositol -3- phosphate synthase (ino) gene knockout Homologous integration frame

The clone of 1.ino Homologous integration fragment

Closed according to the sequences Design of saccharomyces cerevisiae inositol -3- phosphate synthase gene (genbank number of registration is nc_001142) Become primer (primer nucleotide sequences are shown in seq id no.163-168), as shown in table 13.With genome dna as template, use primer Ino_1/ino_6 carry out first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycling first round pcr product is template, is carried out with primer ino_2/ino_3 respectively Nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains inositol -3- phosphate synthase gene 5 '-end dna of the 891bp that length is as shown in seq id no.169 Fragment, reclaims dna fragment using multi-functional dna purification kit, then utilizes restriction enzyme kpn i and bamh i digestion Dna fragment, is then subcloned in the carrier ez-t with identical inscribe ferment treatment, sequence verification obtains, that is, obtain plasmid ez- ino23.

First round pcr product also with primer ino_1/ino_6 is template, is carried out using ino_4/ino_5 Nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains inositol -3- phosphate synthase gene 3 '-end 834bp dna piece that length is as shown in seq id no.170 Section, reclaims dna fragment using multi-functional dna purification kit, then utilizes restriction enzyme bamh i and sac i digestion Dna fragment, is then subcloned in the carrier ez-ino23 with identical inscribe ferment treatment, sequence verification obtains, that is, obtain plasmid ez-ino2345.

The pcr reaction primer of table 13 inositol -3- phosphate synthase gene

2. the structure of the clone of selection markers ade2 and Homologous integration carrier

According to the genotype of host cell w303-1b, choose selection markers ade2 gene as inositol -3- phosphate synthase base Because of the selection markers knocking out, design synthetic primer (primer nucleotide sequences are shown in seq id no.171-174), as shown in table 14, With yeast s288c genome as template, using primer ade2_1/ade2_4 carry out first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle first round pcr product be Template, carried out with primer ade2_2/ade2_3 nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), it is the 2295bp shown in seq id no.175 that amplification obtains length Dna fragment.Reclaim dna fragment using multi-functional dna purification kit, then use restriction endonuclease bamh i and sal i digestion dna piece Section, is then subcloned in the carrier ez-ino2345 with identical inscribe ferment treatment, obtains knocking out inositol -3- phosphate synthase gene Homologous double-crossover integrate frame, sequence verification obtains the dna sequential structure shown in seq id no.14, that is, obtain plasmid ez- ino2345-ade2.

The pcr reaction primer of table 14 selection markers ade2 gene

Embodiment 10 inositol -3- phosphate synthase gene knocks out and Methanogenesis

Gamma glutamyl transpeptidase gene knockout engineered strain w303-1b/g/gt is converted using liac conversion method^-And w303- 1b/gp/gt^-, first with restriction endonuclease not i plasmid ez-ino2345-ade2 linearization process, conversion process is as follows:

(5) competent cell 12000rpm centrifugation 15s, abandons liac..

(8) supernatant is removed in centrifugation, then uses sc trp^-、ade^-Auxotroph culture medium rinses 2 times, then uses sc trp^-ade^- Auxotroph culture medium is resuspended, is applied to sc trp^-ade^-In auxotroph solid medium screening flat board, 30 DEG C of culture 3d. Then picking Colony Culture, is purified its genome dna using Yeast genome dna extracts kit, is carried out using pcr method The identification of knock-out bacterial strain.

(9) (primer nucleotide sequences are shown in seq id to utilize inositol -3- phosphate synthase gene homologous region design synthetic primer No.176-179), as shown in Table 15, using primer ino_t1/ino_t4 carry out first round amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle first round pcr product be Template, carried out with primer ino_t2/ino_t3 respectively nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 2.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), amplification obtains the dna fragment that length is 2412bp, and compares Bacterial strain is only capable of amplifying the dna fragment of 565bp, and result obtains two kinds of works of inositol -3- phosphate synthase gene knockout as shown in Figure 8 Journey bacterial strain w303-1b/g/gt^-ino^-And w303-1b/gp/gt^-ino^-Culture presevation number is cgmcc no.8933).

4. sample every 24h, quantitative analysis is carried out to the glutathione of engineered strain synthesis, test result indicate that in shaking flask Fermentation level culture 72h knocks out inositol -3- phosphate synthase gene engineering bacteria w303-1b/g/gt^-ino^-And w303-1b/gp/gt^- ino^-The total output of glutathione is respectively 655.17mg/l and 980.65mg/l, and the amount of cell exocrine glutathione accounts for 15%, illustrate that inactivating inositol -3- phosphate synthase gene can change membrane structure and promote glutathione to extracellular secretion, Thus improving the yield of glutathione further.

The pcr identification reaction primer that table 15 inositol -3- phosphate synthase gene knocks out

Embodiment 11 integrates high expression yeast gamma-glutamic acid kinases (pro1) and glutathione synthetase (gsh b_m) melt The engineering bacteria of hop protein builds and Methanogenesis

B containing pro1-gsh_mThe Homologous integration vector construction of fusion: according to saccharomyces cerevisiae gamma-glutamic acid kinase gene The sequences Design synthetic primer (primer nucleotide sequences are shown in seq id no.180-183) of (genbank number of registration is m85293), As shown in table 16.With genome dna as template, carried out with primer pro1_1/pro1_4 first round pcr (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1.5min, 30cycles；72 DEG C, 10min；4 DEG C, 10min).Recycle first round pcr product be Template, carried out with primer pro1_2/pro1_3 respectively nested-pcr amplification (95 DEG C, 5min；95 DEG C, 50s, 50 DEG C, 50s, 72 DEG C, 1min, 30cycles；72 DEG C, 10min；4 DEG C, 10min), it is containing as shown in seq id no.9 that amplification obtains length The dna fragment of 1284bp encoder block, i.e. gamma-glutamic acid kinase gene pro1, the amino acid residue sequence of its coding is seq id no.10.Purify and reclaim dna fragment nde i and bamh i digestion, then with the plasmid processing through nde i and bgl ii digestion Carrier tyr14-ug6/t_pgk/p_gap-pc/gsh b_mConnect, conversion Escherichia coli screening obtains positive colony tyr14-ug6/t_pgk/ p_gap-pc/pro1-gsh b_m, that is, obtain the fusion pro1-gsh b being suitable for saccharomyces cerevisiae expression_m, merge therebetween Connection peptide shown in seq id no.12, strengthens the functional domain pro1 and gsh b of two different catalytically actives_mFlexible structure, The two space structure can be made simultaneously neighbouring, improve the efficiency of cascade catalytic reaction.

Table 16 yeast gamma-glutamic acid kinases (pro1) gene pcr reacts primer

Yeast conversion: be integrated with the fusion gsh ii-gsh i of yeast and knock out simultaneously using the conversion of liac conversion method The engineered strain w303-1b/g/gt of gamma glutamyl transpeptidase and inositol -3- phosphate synthase gene^-ino^-1^#(g represents p_gap-sc-gsh ii-gsh i-t_pgkExpression cassette, gt^-Represent gamma glutamyl transpeptidase gene inactivation, ino^-Represent inositol -3- phosphorus Synthase genes inactivate), first with restriction endonuclease not i plasmid tyr14-ug6/t_pgk/p_gap-pc/pro1-gsh b_mDigestion line Property, conversion process is as follows:

(5) competent cell 12000rpm centrifugation 15s, abandons liac..

Positive colony screens: picking positive colony on the ypd flat board of g418, is cultivated using ypd fluid nutrient medium, Purify the conversion subgenom dna of g418 resistance using Yeast genome dna extracts kit, using primer pro1_2/ Gshb2_12 directly carries out pcr reaction identification knock-out bacterial strain, and result negative control as shown in Figure 9 does not have amplified reaction, and obtains Positive colony w303-1b/gp1gt^-ino^-1^#(p1 represents p_gap-pc/pro1-gsh b_m, culture presevation number is cgmcc No.8932 the dna fragment of 2283bp can) be amplified, consistent with control plasmid result.

Glutathione volume analysis: picking positive colony carries out ypd Liquid Culture, is carried out first in shaking flask horizontal analysis paddy The yield of the sweet peptide of Guang:

4. sample every 24h, quantitative analysis is carried out to the glutathione of engineered strain synthesis, test result indicate that in shaking flask Fermentation level cultivates 72h engineering bacteria w303-1b/gp1/gt^-ino^-The total output of glutathione is respectively 1003.45mg/l, and thin The ratio of exocytosis glutathione does not change, and fusion protein pro1-gsh b is described_mExpression and catalysis activity and fusion Albumen prob-gsh b_mCatalytic capability similar.

Embodiment 12 amino acid precursor adds the impact to accumulation glutathione

(1) on picking ypd flat board different phase engineering bacteria w303-1b/g, w303-1b/gp, w303-1b/gp/gt^-、 w303-1b/gp/gt^-ino^-、w303-1b/gp1/gt^-ino^-It is seeded to equipped with 30ml ypd culture with comparison w303b single bacterium colony In the 250ml triangular flask of base, 30 DEG C, 200rpm incubated overnight；

(2) above-mentioned bacterium solution 0.5ml is taken to be transferred in the 250ml triangular flask equipped with 50ml ypd culture medium, 30 DEG C, 200rpm Incubated overnight；

(3) again the bacterium solution of step (2) is transferred in the 500ml triangular flask equipped with 100ml ypd culture medium, controls initial od₆₀₀=0.15, gauze plug seals, 30 DEG C, and 200rpm cultivates；

(4), after culture 48h, experimental group adds the mixed liquor of the glutamic acid, cysteine and glycine of 10ml50mm, makes paddy The final concentration of propylhomoserin, cysteine and glycine is respectively 5mm, and control group then adds the sterilizing ddh of 10ml₂o.

(5) culture 72h sampling, surveys Fungal biodiversity and intracellular glutathione concentrations in experimental group and control group.

Compared with control group, the intracellular gsh yield of experimental group significantly improves, and illustrates that the interpolation of amino acid precursor can be obvious Ground improves the ability of engineering bacteria synthesizing glutathion；But the ratio that different strains improve is different, high expression gamma-glutamic acid is not had to swash The conversion ratio of the comparison of enzyme and engineered strain such as w303-1b/g interpolation amino acid is 23%, and high expression gamma-glutamic acid kinases The conversion ratio that engineered strain adds amino acid improves 75-80% (table 17).Therefore, added by amino acid test result indicate that this A little engineered strains can prepare the bacterial strain of glutathione as further optimize technique biosynthesis.

The different engineered strain of table 17 adds the impact to gsh yield in the horizontal amino acid of shake flask fermentation

It will be apparent to those skilled in the art that can technical scheme as described above and design, make other various Corresponding change and deformation, and all these change and deformation all should belong to the protection domain of the claims in the present invention Within.

Claims

1. a kind of preparation method of the genetic engineering bacterium for synthesizing glutathion, comprising:

I) merge in the intracellular coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase that proceeds to of Host Strains The gene of albumen, and make its height expression；

Ii) in the intracellular base proceeding to coding gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein of Host Strains Cause, and make its height expression；

Iii) knock out the gamma glutamyl transpeptidase gene of Host Strains cell；

Iv) knock out the inositol -3- phosphate synthase gene of Host Strains cell；

Described i), ii), iii), iv) obtain final product the described genetic engineering for synthesizing glutathion with after the completion of random order operation Bacterium；

Described i) and ii) in connect peptide be made up of 6-34 amino acid residue, its formula is: g-x-g-y-g-z, wherein g be sweet Propylhomoserin, x, y, z is respectively glycine, alanine, serine, 1～10 amino acid of threonine, glutamic acid and proline composition Residue；

Described i), ii), iii), iv) in Host Strains be saccharomyces cerevisiae；

Described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein and gamma-glutamic acid The gene of kinases-connection peptide-glutathione synthetase fusion protein starts table by constitutive promoter and/or inducible promoter Reach, described constitutive promoter is any one in gap, pgk, adh1, tef, cyc1, tpi, and described inducible promoter is In gal1, gal2, gal3, gal5, gal7, gal10, cup1, met25 any one；

Described coding gamma glutamyl cysteine synthetase-connection peptide-glutathione synthetase fusion protein and gamma-glutamic acid The gene of kinases-connection peptide-glutathione synthetase fusion protein is by the high expression of integrated mode；Described integrated mode is high Expression is realized by using the integration site delta sequences of the high copy of yeast；

Described gamma glutamyl cysteine synthetase is the amino acid sequence shown in seq id no.2；

Described glutathione synthetase is the amino acid sequence shown in seq id no.4 or seq id no.6；

Described gamma-glutamic acid kinases is the amino acid sequence shown in seq id no.8 or seq id no.10；

The described peptide that connects is the amino acid sequence shown in seq id no.12；

Described gamma glutamyl transpeptidase gene is knocked out by homologous recombination；

Described inositol -3- phosphate synthase gene is knocked out by homologous recombination.

2. preparation method as claimed in claim 1 is it is characterised in that described coding gamma glutamyl cysteine synthetase-company Connect peptide-glutathione synthetase fusion protein and the base of gamma-glutamic acid kinases-connection peptide-glutathione synthetase fusion protein Reason constitutive promoter gap starts expression.

3. preparation method as claimed in claim 1 is it is characterised in that described gamma glutamyl cysteine synthetase is by seq Nucleotide sequence coded shown in id no.1.

4. preparation method as claimed in claim 1 is it is characterised in that shown in described glutathione synthetase seq id no.4 Amino acid sequence nucleotide sequence coded by seq id no.3.

5. preparation method as claimed in claim 1 is it is characterised in that shown in described glutathione synthetase seq id no.6 Amino acid sequence nucleotide sequence coded shown in seq id no.5.

6. preparation method as claimed in claim 1 is it is characterised in that shown in described gamma-glutamic acid kinases seq id no.8 Amino acid sequence is nucleotide sequence coded shown in seq id no.7.

7. preparation method as claimed in claim 1 is it is characterised in that shown in described gamma-glutamic acid kinases seq id no.10 Amino acid sequence nucleotide sequence coded shown in seq id no.9.

8. preparation method as claimed in claim 1 is it is characterised in that nucleosides shown in seq id no.11 for the described connection peptide Sequences code.

9. preparation method as claimed in claim 1 is it is characterised in that described homologous recombination is using as seq id no.13 The homologous double-crossover of shown dna sequence integrates frame.

10. preparation method as claimed in claim 1 is it is characterised in that described homologous recombination is using as seq id The homologous double-crossover of dna sequence shown in no.14 integrates frame.

The genetic engineering bacterium for synthesizing glutathion of the preparation method preparation described in 11. any one of claim 1-10.

12. genetic engineering bacteriums as claimed in claim 11, its deposit number is cgmcc no.8932.

13. genetic engineering bacteriums as claimed in claim 11, its deposit number is cgmcc no.8933.