CN104357416A - Method for modifying protein folding secretion pathway to enhance GOD (glucose oxidase) secretion - Google Patents

Method for modifying protein folding secretion pathway to enhance GOD (glucose oxidase) secretion Download PDF

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CN104357416A
CN104357416A CN201410568530.6A CN201410568530A CN104357416A CN 104357416 A CN104357416 A CN 104357416A CN 201410568530 A CN201410568530 A CN 201410568530A CN 104357416 A CN104357416 A CN 104357416A
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god
glucose oxidase
pichia pastoris
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张娟
陈坚
顾磊
堵国成
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Jiangnan University
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

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Abstract

The invention discloses a method for modifying a protein folding secretion pathway to enhance GOD (glucose oxidase) secretion and belongs to the technical field of genetic engineering. According to the method, genes such as KAR2, CNE1, ERO1, SSA4, SSO2, SEC53, BMH2, HRD1, UBC1 and GCN4 in Pichia pastoris are cloned and connected to a series of derivative plasmids such as a Pichia pastoris expression vector pGAPZ by a gene recombination technology, the genes and GOD are co-expressed and transformed into a Pichia pastoris GS115 strain, the effect is inspected, modular combination optimization is conducted, and a strain, which enhances GOD secretory expression as compared with the original strain, is obtained by screening and identification. The GOD expressed by the strain achieves the enzyme activity of 1,972.9 U/mL on a 3L fermentation tank. The method lays a favorable foundation for large-scale production of GOD.

Description

A kind of engineered protein folds the method that Secretory Pathway strengthens Glucose oxidase secretion
Technical field
The present invention relates to a kind of engineered protein and fold the method that Secretory Pathway strengthens Glucose oxidase secretion, belong to gene engineering technology field.
Background technology
Glucose oxidase is one of topmost toolenzyme in biological field.From Updike and Hicks in 1967, GOD is fixed on Clark oxygen electrode surface, since being applied to blood sugar detection, GOD is widely used in many association areas such as food, feed, medicine.
In the food industry, due to the existence of oxygen, cause many chemical reactions being unfavorable for quality product, and create condition for many microorganism growth.At present, GOD is widely used in various food and food processing technology as the safe oxidation inhibitor of workman by many countries.Although purposes is various, the effect of GOD is mainly except glucose, deoxygenation, formation hydrogen peroxide, forms gluconic acid four aspects.Utilize its single-minded oxidasic principle, make glucose oxidase assay instrument, quick and precisely can measure the glucose content in various food simply, Instructing manufacture.
In medicine industry, GOD is as the Quantitative in vitro analysis for glucose in serum (slurry), urine and cerebrospinal fluid such as test kit, enzyme electrodes; The zymin that GOD makes also can be used for removing or alleviate the generation that the formation of dental plaque, tartar and carious tooth prevents oral disease and odontopathy.In addition, due to catalysis H202 can be generated, also can be used for the treatment of the lymphadenomatous target goal to H2O2 sensitivity.
GOD or a kind of novel enzyme feed additive, can improve animal intestinal environment, regulates diet digestion, promote growth of animal.Containing the mixed fodder additive of glucose oxidase, lactic acid superoxide and lactoferrin, can be used for the gastrointestinal tract infection of prevention livestock, diarrhoea, and have the effect of promotion growth of animal.
From animal vegetable tissue, extract GOD have certain limitation, enzyme amount is not also enriched; Bacterium GOD yield of enzyme is few; General employing aspergillus niger (having GRAS qualification) and Penicillium bacterial strain produce bacterium as GOD.China and the U.S. all adopt a mould and Penicllium chrysogenum to produce GOD, the conventional rugged mould of Buddhist nun of Japan, Russia uses life mould, in recent years reports that the mould genus of glue (Clioctadium), paecilomyces (Paecilomyces) and the mould genus of broom (Scopulariopsis) also can produce GOD.
Yield poorly, enzyme live low, detection method complicated be the restrictive factor of GOD industrialization, do a lot of work both at home and abroad for this reason and achieve obvious progress.The external GOD producer produced the mainly Boehringer of Germany and the TOYOBO of Japan at present.The highly active GOD of large-scale production also has any problem.Produce a large amount of foreign protein while fermentative production GOD, separation and Extraction is complicated, and cost is high.
Utilize genetically engineered or fermentation engineering to study the overexpression of GOD.The suitability for industrialized production that its zymologic property, molecular modification finally realize glucose oxidase has established certain theoretical basis and strategy guiding.
Summary of the invention
The invention provides a kind of engineered protein and fold the method that secretion path strengthens Glucose oxidase secretion, express on the basis of glucose oxidase gene GOD in Pichia pastoris GS115, further the genes such as KAR2, CNE1, ERO1, SSA4, SSO2, SEC53, BMH2, HRD1, UBC1, GCN4 in pichia spp are cloned series derivatives plasmids such as being connected to yeast expression vector pGAPZ respectively and carry out coexpression, or modular combination expression is carried out to said gene, obtains Glucose oxidase secretion and express the bacterial strain strengthened.
Described engineered protein folds secretion path and strengthens in the method for Glucose oxidase secretion, and protein folding secretion path comprises three modules, and wherein, module one comprises vesicle transport gene SSA4, SSO2, SEC53, BMH2, ERAD gene HRD1, UBC1; Module two comprises folding gene KAR2, CNE1, ERO1 in endoplasmic reticulum; Module three coerces stress response gene GCN4; Described transformation is any one gene in expression three modules, or any one gene in any one gene of simultaneously expressing in module one and module two, or expresses each gene in module one to three simultaneously.
In one embodiment of the invention, the nucleotide sequence of SSA4 gene is as shown in Gene ID:8199979, the nucleotide sequence of SSO2 gene is as shown in Gene ID:8197744, the nucleotide sequence of SEC53 gene is as Gene ID:8199158, the nucleotide sequence of BMH2 gene is as shown in Gene ID:8198501, the nucleotide sequence of HRD1 gene is as Gene ID:8201066, the nucleotide sequence of UBC1 gene is as shown in Gene ID:8200711, the nucleotide sequence of KAR2 gene is as shown in Gene ID:8198455, the nucleotide sequence of CNE1 gene is as shown in Gene ID:8198102, the nucleotide sequence of GCN4 gene is as shown in Gene ID:8197788.
The present invention also provides a kind of Glucose oxidase secretion to express the recombinant yeast pichia pastoris strengthened, and is on the basis of expressing glucose oxidase gene, has transformed protein folding secretion path.Described protein folding secretion path comprises three modules, and wherein, module one comprises vesicle transport gene SSA4, SSO2, SEC53, BMH2, ERAD gene HRD1, UBC1; Module two comprises folding gene KAR2, CNE1, ERO1 in gene endoplasmic reticulum; Module three coerces stress response gene GCN4; Described transformation is any one gene in expression three modules, or expresses each gene in module one, two simultaneously, or expresses each gene in module one to three simultaneously.
Described recombinant yeast pichia pastoris, with yeast expression vector pGAPZ α A plasmid for expression vector expressing protein folding secretion pathway gene; To remove HIS4 and kan rpPIC9k express glucose oxidase gene.
The pGAPZ that the α-mating signal peptide that described pGAPZ plasmid comprises removal pGAPZ α A obtains, remove the α-mating signal peptide of pGAPZ α A, and Shble is replaced to the pGAPH that HIS4 obtains, remove the α-mating signal peptide of pGAPZ α A, and Shble is replaced to kan rthe pGAPK obtained.
In one embodiment of the invention, express the gene of module one with plasmid pGAPZ, express the gene of module two with plasmid pGAPH, express the gene of module three with plasmid pGAPK.
In one embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression endoplasmic reticulum gene C NE1 or vesicle transport gene SEC53 or ERAD gene HRD1 or coerce stress response gene GCN4.
In another embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression module one SEC53 gene and module two KAR2 gene.
In another embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression module one SSA4 gene and module two CNE1 gene.
In another embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression module one SEC53 gene and module two CNE1 gene.
In another embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression module one SEC53 gene, module two CNE1 gene and module three GCN4 gene.
In another embodiment of the invention, in P.pastoris GS115, express glucose oxidase gene GOD, and coexpression module one SEC53 gene, module two KAR2 gene and module three GCN4 gene.
The present invention also provides a kind of and applies the method that described recombinant yeast pichia pastoris produces glucose oxidase, cultivates OD by under recombinant bacterium 30 DEG C, 200rpm 600seed between 1.6 ~ 1.7 proceeds to basic fermention medium (BMGY) with the inoculum size of 2%, in 30 DEG C, cultivate under 200rpm condition; OD is cultured in basic fermention medium 600when value is for 1.2-1.5, yeast cell is proceeded to the generation of inducible protein in inducing culture (BMMY).
In one embodiment of the invention, the method that described recombinant bacterium produces glucose oxidase is applied in 3L fermentor tank, comprise the following steps: be in 10% aseptic technique access 3L automatic fermenter (LiFlus GM BioTRON, Korea) by the recombinant bacterium bacterium liquid activated with inoculum size; Starting condition is: liquid amount 800-1000mL, initial mixing speed is 500r/min, air flow 2.5vvm, phosphoric acid solution with 30% and 25% strong aqua control pH be 5.5, the culture temperature in vegetative period is 30 DEG C, adopt the DO-stat stirring association to control, maintain DO 30%, mixing speed maximum is set to 950r/min; When glycerol depletion (DO rises rapidly), during DO>60%, start index stream adds the glycerin medium of interpolation 50%; Treat that glycerine exhausts again, DO rebounds again, work as DO>60%, start induction, inducing temperature is reduced to 22 DEG C, start stream and add inducing culture, make substratum methanol concentration reach rapidly 1.8% (w/w), adding controller control methyl alcohol residual concentration by FC2002 type methyl alcohol detection stream is 1.8% (w/w).
Shown basic fermention medium is BMGY substratum (1L): Tryptones 20g, yeast extract 10g, glycerine 10mL, YNB13.4g, 100mM phosphoric acid buffer (pH6.0); Inducing culture is BMMY substratum (1L): Tryptones 20g, yeast extract 10g, methyl alcohol 8mL, YNB13.4g, 100mM phosphoric acid buffer (pH6.0).
The present invention adopts gene recombination technology the genes such as KAR2, CNE1, ERO1, SSA4, SSO2, SEC53, BMH2, HRD1, UBC1, GCN4 in pichia spp to be cloned respectively the connection such as the series derivatives plasmid that is connected to yeast expression vector pGAPZ, and be transformed in Pichia pastoris GS115 bacterial strain with GOD coexpression, obtain the recombinant bacterium that a series of Glucose oxidase secretion strengthens; And optimized by modular combination, obtain the bacterial strain that the more original bacterial strain of a strain strengthens secreting, expressing glucose oxidase, enzyme 1972.9U/mL alive on 3L fermentor tank.The present invention is that the scale operation of glucose oxidase is had laid a good foundation.
Accompanying drawing explanation
Fig. 1 functional module 1 and module 2 assortment of genes optimization are on the impact of heterogenous expression GOD; (A) GOD extracellular protein content; (B) GOD born of the same parents are outer active; (C) dry cell weight DCW.
Fig. 2 functional module 1 and module 2 assortment of genes are on the impact of producing GOD production efficiency.
Fig. 3 functional module 3 assortment of genes is on the impact of producing GOD production efficiency.
ROS level and cell survival rate in different strains born of the same parents in Fig. 4 fermenting process; A, cell survival rate, ■ GS115, PP-GOD, ● S17; B, high ROS horizontal cell amount; The horizontal dead cell amount of C, high ROS; gS115; pP-GOD; s17.
Embodiment
Table 1 the present invention builds bacterial strain and plasmid
Table 2 the present invention builds plasmid the primer
1)in design primer sequence, underlined letter representative designs the restriction enzyme site added.
Glucose oxidase enzyme activity determination method: GOD determination of activity generally adopts o-(two) methyl oxyaniline spectrophotometry.Under aerobic conditions, GOD catalysis glucose dehydro produces H 2o 2, under peroxidase (POD) effect, oxygen donor o-(two) methyl oxyaniline (DH 2) be oxidized to brown product.Survey the change of 540nm place absorbancy, according to the result of typical curve, calculate glucose oxidase enzyme activity unit.
Seed and slant medium are YPD substratum (1L): Tryptones 20g, yeast extract 10g, glucose 20g; Slant medium adds agar 20g.
Basic fermention medium is BMGY substratum (1L): Tryptones 20g, yeast extract 10g, glycerine 10mL, YNB13.4g, 100mM phosphoric acid buffer (pH6.0).
Inducing culture is BMMY substratum (1L): Tryptones 20g, yeast extract 10g, methyl alcohol 8mL, YNB13.4g, 100mM phosphoric acid buffer (pH6.0).
The variant module gene of embodiment 1 is on the impact of secretion glucose oxidase
The impact of folding module gene pairs heterogenous expression GOD in 1.1 transformation endoplasmic reticulum
Protein folding speed in endoplasmic reticulum (ER) and intracytoplasmic secreting rate are the important factors affecting heterologous protein secretion in yeast cell.Resident albumen Kar2p, Cne1p and Ero1p that participate in protein folding mechanism in ER can help the not folding peptide chain of new life to be folded into correct conformation.The impact of module on secretion external source GOD is folded in order to study ER, by resident for the ER in P.pastoris protein coding gene KAR2, CNE1 and ERO1 is respectively at the excessive coexpression of P.pastoris engineering bacteria PP-GOD, to strengthen the expression level of the corresponding function albumen in improvement project strain cell, result is as shown in table 3.In improvement project bacterium PP-G-CNE1 after coexpression GOD and CNE1, GOD activity reaches 779.3UmL -1, average foreign protein productivity qp and GOD productivity Qp reaches 0.528mgg simultaneously dCW -1h -1and 35.48Ug dCW -1h- 1.Compare control strain PP-GOD to significantly improve.This finds that coexpression CNE1 gene can improve the secretion of external source GOD in P.pastoris first time.CNE1 gene plays an important role for folding in ER of glycated protein in yeast cell, and it can strengthen the folding of new polypeptide chain, thus improves output.
But not all module gene is all very remarkable for the effect strengthening secretion external source GOD.In improvement project bacterium PP-G-KAR2, less on statistical significant difference, active in relative to control strain PP-GOD, GOD 322.9UmL -1bring up to 378.1UmL -1.The specific production rate (GOD protein content/DCW) of GOD is in a slight decrease simultaneously, from 0.046gg dCW -1be reduced to 0.039gg dCW -1.Same in improvement project bacterium PP-G-ERO1, coexpression ERO1, remarkable on the impact of secretion external source GOD, reach 347.2UmL compared to control strain PP-GOD, GOD activity and average protein production rate qp -1and 0.486mgg dCW -1h -1, promote significant difference less.
The impact of folding module gene pairs heterogenous expression GOD in table 3 pichia spp
1.2 transformation vesicle transport module genes are on the impact of heterogenous expression GOD
In foreign protein Secretory Pathway, between different organoid, the transhipment of albumen is by solvable SNARE synaptic membrane protein acceptor (N ethylmaleimide sensitive factor receptor) catalysis and regulation and control.Although foreign protein has defined correct conformation in yeast host cell secretion process, it has been caused to be accumulated in born of the same parents because vesicle transport efficiency is not high.Cause secretion effect inapparent trafficking step, contain kytoplasm to ER, ER to golgi body, kytoplasm is to different stage such as cytolemma.In order to study the impact of vesicle transport module for secretion external source GOD, by the encoding gene SSA4 in pichia spp, SSO2, SEC53 and BMH2 are at the excessive coexpression of Pichia yeast engineering PP-GOD, and result is as shown in table 4.Transport gene location SSA4 in process LAN kytoplasm to ER in transformation bacterial strain PP-G-SSA4 after, GOD activity reaches 412.7UmL -1, average GOD productivity Qp reaches 22.46Ug dCW -1h -1, simultaneously average protein productivity qp has compared to control strain PP-GOD and promotes by a small margin, from 0.431mgg dCW -1h -1bring up to 0.459mgg dCW -1h -1.In the PP-G-BMH2 of coexpression BMH2 gene, the reinforced effects for secretion external source GOD is general, and average qp and Qp reaches 0.462mgg respectively dCW -1h -1and 20.39Ug dCW -1h -1.The effect that coexpression SSO2 compares SSA4 and BMH2 is better, and average qp and Qp brings up to 0.510mgg dCW -1h -1and 26.41Ug dCW -1h -118% and 52% is improve respectively compared to control strain PP-GOD, this illustrates the effect of syntaxin acceptor Sso2p for P.pastoris intracellular vesicle transhipment external source GOD, enters ER and leave the impact of transhipment step on secretion external source GOD entering kytoplasm and cytolemma from ER larger compared to kytoplasm.
In addition, the transformation bacterial strain PP-G-SEC53 of coexpression SEC53 gene is to the Be very effective of secretion external source GOD.Compared to control strain PP-GOD, average Qp and qp is all improved largely, respectively from 17.43Ug dCW -1h -1be increased to 31.58Ug dCW -1h -1, 0.431mgg dCW -1h -1be increased to 0.545mgg dCW -1h -1, the outer GOD activity of final born of the same parents is also from 322.9UmL simultaneously -1be increased to 960.4UmL -1, improve nearly 1.9 times.This illustrates that coexpression SEC53 gene can reduce the folding pressure of ER inside by improving the exogenous sugar albumen efficiency that glycosylation controls in ER, thus improves the secretion of external source GOD.
In table 4 pichia spp, vesicle transport module gene is on the impact of heterogenous expression GOD
1.3 transformation ERAD module genes are on the impact of heterogenous expression GOD
Overexpression external source GOD can increase the weight of the load pressure of ER.When the foreign protein of the not folding peptide chain be retained in ER and folding mistake accumulates in a large number, the time forming correct conformation can be made to increase, thus form coercing ER.Overload and the albumen of wrong conformation proceed in kytoplasm by ERAD mechanism, and guiding enters proteasome (Proteasome) and degrades further, and that effectively can slow down ER coerces pressure.Ubiquitin ligase in HRD1 genes encoding ERAD mechanism is the important participation factors of degraded path.In table 5, as the transformation bacterial strain PP-G-HRD1 of coexpression HRD1 gene, increase substantially GOD output, reached 837.7UmL -1.Average Qp and qp also obviously promotes simultaneously, brings up to 32.86Ug respectively dCW -1h -1and 0.524mgg dCW -1h -1, improve 88% and 21% respectively compared to control strain.The final growing state of host cell is also clearly better, and final DCW is increased to 271.7gL -1.
In table 5 pichia spp, ERAD module gene is on the impact of heterogenous expression GOD
Compare coexpression HRD1 gene, as participating in the ubiquitin conjugation enzyme coding gene UBC1 screening the improper albumen be degraded in ERAD mechanism, in coexpression transformation bacterial strain PP-G-UBC1, smaller on statistical significant difference, average Qp and qp promotes not remarkable, and comparatively control strain only improves 17% and 16% respectively.In ER, the prolonged stay of paraprotein can cause the metabolism of ERAD mechanism to bear, and can cause serious impact to host, and what directly cause is exactly weakening of thalli growth ability.The major function gene of result display coexpression ERAD module can provide the help of forward for secreting external source GOD, can improve the energy for growth of cell by slowing down ERAD Metabolic stress simultaneously.
The impact of stress reaction module gene on heterogenous expression GOD is coerced in 1.4 transformations
Based on above-mentioned result, find that coexpression ERAD module important gene can improve the secretion production of external source GOD, also can improve the energy for growth of host cell when overexpression foreign protein simultaneously.These results also demonstrate that dysfunction and the retroaction of Growth of Cells, and the secretion for secretion external source GOD is produced serious impact.When excessive secretion foreign protein, the dysfunction of Growth of Cells causes mainly due to the pressure of coercing in ER.When coercing pressure in ER and occurring, the stress reaction regulatory factor existed as the mechanism resisted in born of the same parents can be activated, wherein Gcn4p is UPR stress reaction incitant, and Gcn4p is different from HAC1 genes encoding regulatory factor, and the main maintenance cell function that participates in resists growth dysfunction.
Coerce in table 6 pichia spp stress module gene on the impact of heterogenous expression GOD
In order to verify that coerce stress the impact of module gene GCN4 gene pairs secretion external source GOD, GCN4 gene is carried out coexpression in transformation bacterial strain PP-G-GCN4.The results are shown in Table 6, cell growth status comparatively control strain PP-GOD has clear improvement, and final DCW is from 134.4gL -1be increased to 288.8gL -1, improve 1.1 times.GOD activity significantly promotes simultaneously, reaches 1239.6UmL -1, average Qp and qp promotes and also significantly promotes.
Embodiment 2 protein secretory pathway module optimizes the impact on heterogenous expression glucose oxidase
Embodiment 1 is each module gene of coexpression respectively, can improve the output of GOD in restructuring P.pastoris, this imply that modules transformation P.pastoris secretes external source GOD and all has effect.Produce the ability of external source GOD to improve restructuring P.pastoris further, the optimum combination of research modules gene is on the impact of producing external source GOD ability.Due to connecting each other between each mac function, the functional module gene in a simple superposition mac function, can not make result present the increase of superposing type.Therefore, the gene of difference in functionality module, by the mode of modularization Optimizing Reconstruction, combines by the present embodiment mutually, produces external source GOD to improve restructuring P.pastoris further.Functional gene in embodiment 1 is divided three classes, carry out ensuing Combinatorial Optimization, be respectively: (1) module 1: contain HRD1, SEC53, SSO2 and SSA4 gene, gene in module 1 contains the gene of ERAD functional module and born of the same parents' intracellular vesicle transportation module, can slow down the accumulated pressure of albumen in born of the same parents in the organoid such as endoplasmic reticulum, strengthen transportcapacity; (2) module 2: comprise KAR2, CNE1 and ERO1, the gene in module 2 is folding module gene in endoplasmic reticulum, directly affects the Folding rate of albumen in endoplasmic reticulum; (3) module 3: contain GCN4 gene, the module 3 genes encoding factor belongs in born of the same parents the stress reaction regulatory factor that the mechanism resisted exists, and can affect anti-the coerce ability of P.pastoris to stress conditions in born of the same parents.
The Combinatorial Optimization of 2.1 functional modules 1 and module 2 is on the impact of heterogenous expression GOD
For the impact of producing external source GOD, combination coexpression is carried out to the typical encoding gene of module 1 and module 2, as shown in Figure 1 in order to investigate Folding rate and transmitter loss ability in endoplasmic reticulum ER.
When the gene of module 2 is KAR2, module 1 is respectively HRD1, and SEC53, SSO2 and SSA4 gene and KAR2 carry out collocation coexpression.When the KAR2 in the SEC53 in module 1 and module 2 arranges in pairs or groups coexpression, the external source GOD output of this recombinant bacterium is the highest, and the outer content of GOD born of the same parents is up to 16.09gL -1, the outer activity of GOD born of the same parents reaches 1305.7UmL -1, dry cell weight reaches 361.8gL simultaneously -1.
When the gene of module 2 is CNE1, in module 1, HRD1, SEC53, SSO2 and SSA4 gene carries out collocation coexpression with it respectively.The second external source GOD output of taking turns middle different recombinant bacterium presents identical trend with the optimization of the first round, SEC53 gene carries out collocation coexpression with gene in folding module 1 can reach best effect, this illustrates involved in sugar albumen glycosylation modified SEC53 gene controlling transhipment in endoplasmic reticulum, has special effect for Glycoprotein G OD enhancing of Folding rate in endoplasmic reticulum.In addition, this result of taking turns demonstrates the advantage of coexpression CNE1 gene compared with KAR2 gene, and the outer content of GOD born of the same parents of this recombinant bacterium is up to 17.43gL -1, the outer activity of GOD born of the same parents reaches 1559.4UmL -1.
When the gene of module 2 is ERO1, find that the comparatively front two-wheeled effect of optimization of effect is obvious.
As shown in Figure 2, average Qp and qp of the middle GOD of restructuring P.pastoris bacterial strain (S2, S4, S6, S8, S10 and S12) of result display coexpression SEC53 or SSA4 does not have the recombinant bacterial strain of these two genes of coexpression higher compared with other.Describe in endoplasmic reticulum the function and transhipment positioning function that participate in glycosylation control, have important effect to the secretion of raising external source GOD in host cell.Meanwhile, average Qp and qp of recombinant bacterial strain (S2, S4, S6 and S8), under the condition of coexpression KAR2 and CNE1 gene, wants high under not expressing their condition compared with other.Recombinant bacterial strain S2, during coexpression SEC53 and KAR2, average Qp and qp promotes the most obvious at the same time, and Qp reaches 54.10Ug dCW -1h -1, qp is up to 0.735mgg simultaneously dCW -1h -1.
The Combinatorial Optimization of 2.2 functional modules 3 is on the impact of heterogenous expression GOD
The Combinatorial Optimization of functional module 1 and functional module 2, finds that the intergenic collocation of HRD1, SEC53, SSA4, KAR2 and CNE1 has heterogenous expression GOD and comparatively significantly acts on.Therefore, on this basis, functional module 3 is carried out to the investigation of Combinatorial Optimization.Take turns in Combinatorial Optimization at this, the coexpression array mode of module 1 and module 2 at bacterial strain S1, on the combination foundation of S2, S4, S5, S6 and S8, the optimization of laminating module 3, as shown in Figure 3.Result display in figure, after coexpression module 3 gene GCN4, can improve gathering of the outer GOD of born of the same parents effectively.The GOD output increased wherein transforming recombinant bacterial strain S17 is the most remarkable, when while coexpression SEC53, during CNE1 and GCN4 gene, GOD activity is up to 1972.9UmL -1, average qp is up to 0.971mgg dCW -1h -1.
The impact of embodiment 3 modularization transformation on pichia spp intracellular reactive oxygen species generation level
The changing conditions of ROS and Growth of Cells in modified recombinant bacterium S17 born of the same parents has been investigated by flow cytometer.Owing to utilizing methyl alcohol also can have an impact to the accumulation of ROS in born of the same parents, therefore simultaneously by wild-type GS115 bacterial strain in contrast bacterial strain compare together.As shown in Figure 4, through transformation, the cell survival rate of S17 bacterial strain significantly promotes, and fermentation 144h cell survival rate reaches 77.4%, compares survival rate improve 16.6% with original strain PP-GOD.By the detection to ROS level in born of the same parents, find owing to adopting methanol induction, thalline also can produce ROS and improve the level of ROS in born of the same parents while utilizing methyl alcohol, as shown in Fig. 4-B, control strain GS115 is along with the prolongation of induction time, the cell count of high ROS level is also in increase, and after inducing 96h, the cell count of high ROS level accounts for 76% of total cell count; But find in bacterial strain PP-GOD and the S17 of overexpression GOD, because overexpression heterologous protein GOD can make folder function in ER enter full load condition, also can produce ROS thus cause oxidative stress, its high ROS horizontal cell number ratio is obviously higher than control strain, ascendant trend is also more obvious, and 48h is just close to 70% in induction.Illustrate that overexpression GOD causes extra ROS burden to cell.This demonstrate that the accumulation of ROS can affect saccharomycetic growth really, overexpression foreign protein can cause extra ROS accumulation simultaneously.As shown in Fig. 4-C, to the comparison of the horizontal dead cell ratio of ROS high in each bacterial strain, find that in original recombinant bacterium PP-GOD, high ROS horizontal dead cell ratio is higher, and the dead cell ratio transforming bacterium S17 obviously declines, illustrate that Reconstruc-tion policy in S17 bacterial strain is while more High-efficient Production GOD, the ability of anti-ROS also significantly strengthens, and cell survival rate is improved, thus further increases throughput.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. strengthen a method for Glucose oxidase secretion, be express on the basis of glucose oxidase gene GOD in pichia spp (Pichia pastoris), further engineered protein folds Secretory Pathway; Described protein folding Secretory Pathway comprises three modules, and wherein, module one comprises vesicle transport gene SSA4, SSO2, SEC53, BMH2, ERAD gene HRD1, UBC1; Module two comprises folding gene KAR2, CNE1, ERO1 in endoplasmic reticulum; Module three coerces stress response gene GCN4; Described transformation is any one gene in expression three modules, or any one gene in any one gene of simultaneously expressing in module one and module two, or expresses each gene in module one to three simultaneously.
2. method according to claim 1, it is characterized in that, the nucleotide sequence of SSA4 gene is as shown in Gene ID:8199979, the nucleotide sequence of SSO2 gene is as shown in Gene ID:8197744, the nucleotide sequence of SEC53 gene is as GeneID:8199158, the nucleotide sequence of BMH2 gene is as shown in Gene ID:8198501, the nucleotide sequence of HRD1 gene is as Gene ID:8201066, the nucleotide sequence of UBC1 gene is as shown in Gene ID:8200711, the nucleotide sequence of KAR2 gene is as shown in Gene ID:8198455, the nucleotide sequence of CNE1 gene is as shown in Gene ID:8198102, the nucleotide sequence of GCN4 gene is as shown in Gene ID:8197788.
3. a Glucose oxidase secretion expresses the recombinant yeast pichia pastoris strengthened, on the basis of expressing glucose oxidase gene, transform protein folding Secretory Pathway, described protein folding Secretory Pathway comprises three modules, wherein, module one comprises vesicle transport gene SSA4, SSO2, SEC53, BMH2, ERAD gene HRD1, UBC1; Module two comprises folding gene KAR2, CNE1, ERO1 in gene endoplasmic reticulum; Module three coerces stress response gene GCN4; Described transformation is any one gene in expression three modules, or expresses each gene in module one, two simultaneously, or expresses each gene in module one to three simultaneously.
4. recombinant yeast pichia pastoris according to claim 3, is characterized in that, with pGAPZ α A plasmid for expression vector expressing protein folds Secretory Pathway gene; To remove HIS4 and kan rpPIC9k express glucose oxidase gene.
5. recombinant yeast pichia pastoris according to claim 3, it is characterized in that, the pGAPZ that the α-mating signal peptide that described pGAPZ α A plasmid comprises removal pGAPZ α A obtains, remove the α-mating signal peptide of pGAPZ α A and Shble replaced to the pGAPH that HIS4 obtains, remove the α-mating signal peptide of pGAPZ α A and Shble is replaced to kan rthe pGAPK obtained.
6. recombinant yeast pichia pastoris according to claim 5, is characterized in that, expresses the gene of module one with plasmid pGAPZ, expresses the gene of module two with plasmid pGAPH, expresses the gene of module three with plasmid pGAPK.
7. recombinant yeast pichia pastoris according to claim 3, it is characterized in that, in P.pastorisGS115, express glucose oxidase gene GOD, and coexpression endoplasmic reticulum gene C NE1 or vesicle transport gene SEC53 or ERAD gene HRD1 or coerce stress response gene GCN4.
8. recombinant yeast pichia pastoris according to claim 3, is characterized in that, is to express glucose oxidase gene GOD in P.pastoris GS115, and coexpression module one SEC53 gene and module two KAR2 gene.
9. recombinant yeast pichia pastoris according to claim 3, is characterized in that, expresses glucose oxidase gene GOD in P.pastoris GS115, and coexpression module one SEC53 gene, module two CNE1 gene and module three GCN4 gene.
10. the application of recombinant yeast pichia pastoris according to claim 3 in glucose oxidase is produced.
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