CN103088052A - Carrier for promoting expression of phytase in pichia pastoris - Google Patents
Carrier for promoting expression of phytase in pichia pastoris Download PDFInfo
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- CN103088052A CN103088052A CN2012105500264A CN201210550026A CN103088052A CN 103088052 A CN103088052 A CN 103088052A CN 2012105500264 A CN2012105500264 A CN 2012105500264A CN 201210550026 A CN201210550026 A CN 201210550026A CN 103088052 A CN103088052 A CN 103088052A
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Abstract
The invention relates to a carrier for promoting expression of phytase in pichia pastoris, belongs to the technical field of microbial engineering, and particularly relates to a carrier containing a translation regulatory factor Hacl. The expression level of the phytase of engineering bacteria is obviously improved after the carrier is led to the pichia engineering bacterium of expressing the phytase. The pichia engineering bacterium has the advantages of extracellular secretion and posttranslational modification. Therefore, the super-level expression pichia engineering bacterium can be applied to industrial production; and the fermentation cost is saved.
Description
Technical field
The present invention relates to the microbial engineering field, be specifically related to a kind of Pichia yeast engineering that contains the expression vector of translational control factor Hac1 and contain this carrier.
Background technology
The heterogenous expression of protein is one of emphasis of biology field research commonly used; Particularly for suitability for industrialized production, expressing over one's competence is to enhance productivity and one of cost-effective key means.The host of protein heterogenous expression is a lot, and prokaryotic micro-organisms is arranged, as intestinal bacteria and subtilis etc.; Eukaryotic microorganisms is also arranged, as pichia spp and Trichodermareesei etc.Prokaryotic organism have for the time short, the fast advantage of growing; But go to lack in born of the same parents the posttranslational modification system.Therefore, procaryotic heterogenous expression albumen does not often have activity, in other words expression level very low (Zhang Xiaoxia etc., 2004, foreign medical science hygiology fascicle).And eukaryotic microorganisms has the posttranslational modification system, often has again the advantage of secreting, expressing and high density fermentation simultaneously; Therefore, increasing eukaryotic microorganisms is developed to be applied to manufacture enzyme and enzyme food etc.No matter be academic research or large-scale industrial production, pichia spp (Pichia pastoris) is one of the most frequently used eukaryotic expression system (Porro etc., 2005, Mol. Biotechnol.).
But the protein expression level of pichia spp is very low, and the target protein content of most engineering bacteria exocytosiss is about 8g/L(Niebaur etc., 2005, Protein expression technologies).There is several factors to affect protein expression level, as promotor, copy number, signal peptide, codon bias, (Payne etc. 2008, Appl. Environ. Microbiol.) such as translation modification and zymotechniques.But a lot of promotion protein foldings in the pichia spp born of the same parents are found in research, and the factor of vesica transportation and cross-film transportation can significantly improve the protein expression level.
Only have correct folding protein just can be transported to outside born of the same parents.If can not correctly fold after the foreign protein translation, can cause the non-correct response of albumen, be called for short UPR reaction (unfolded protein response is called for short UPR).UPR can suppress transcribe and react; Promote simultaneously the degraded of misfolded protein matter, thereby affect protein expression level (Alimjan, 2010, Appl Microbiol Biotechnol).The UPR reaction can regulate and control the expression of a lot of in born of the same parents and protein expression correlation factor, as molecular chaperones, (the Mouna such as the kinases that folding enzymes, vesica transportation are correlated with and transcription factor Hac1P, 2010, Microbial Cell Factories), wherein Hac1P can promote protein expression (Saloheimo, 2003, Mol.Microbiol).The expression of Hac1P is subject to the strict regulation and control of UPR.Hac1-mRNA is constitutive expression, but 1 intron is contained in its inside; After only having intron to be sheared, Hac1-mRNA could correction.
Summary of the invention
The purpose of this invention is to provide a kind of expression vector that contains translational control factor Hac1, and obtain new engineering bacteria by this expression vector being imported the Pichia yeast engineering that produces source of phytase, building.In described engineering bacteria, the expression level of source of phytase is significantly improved, and is with a wide range of applications.
One aspect of the present invention provides a kind of carrier for expression of eukaryon for expressing translational control factor Hac1 gene.
The nucleotides sequence of above-mentioned translational control factor Hac1 gene is classified SEQ ID NO:2 as.
The aminoacid sequence of above-mentioned translational control factor Hac1 genes encoding is SEQ ID NO:3.
Above-mentioned expression vector is Pichia anomala expression plasmid pGAPZ.
The present invention also provides a kind of method that phytase is expressed that improves in Pichia yeast engineering, be that above-mentioned expression vector is changed in the Pichia yeast engineering of Expressing Recombinant Phytase, forms new recombinant bacterial strain.
Above-mentioned recombinant bacterial strain is pichia spp SDLH-1(Pichia pastoris SDLH-1), in on December 5th, 2012 be preserved in be positioned at Lopa Nationality an ancient woman's ornament mountain, Wuhan Wuhan University " Chinese Typical Representative culture collection " center ", preserving number are CCTCC NO:M 2012503.
The present invention also provides the application of above-mentioned Pichia yeast engineering SDLH-1, is used for high efficient expression source of phytase.
The present invention has built an expression vector of expressing translational control factor Hac1 gene, simultaneously this carrier is imported the Pichia yeast engineering of phytase generating, obtains new engineering bacteria.The level of this project bacterium Expressing Recombinant Phytase is significantly improved, and expression amount improves more than 17%.
Description of drawings
Fig. 1: the plasmid genetic map of the carrier for expression of eukaryon that is used for expression translational control factor Hac1 gene that the present invention builds.
Embodiment
Following examples are to set forth content of the present invention for explanation better, and the relevant technician in this area can understand better and grasp the present invention by embodiment.But, the case that protection of the present invention and claim scope are not limited to provide.
Embodiment 1:Hac1 gene cloning
1.1 the extraction of total DNA
With the pichia spp incubated overnight, get appropriate thalline and be placed in centrifuge tube, centrifugal 5 min of 13000 rpm abandon supernatant; Add 400 μ l extraction buffers (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl, 1%SDS); Then 95 ℃ of heating 5min add 100mg quartz sand or granulated glass sphere, beat instrument thermal agitation 2min left and right on pearl; After 65 ℃ of water-bath 20min, add 200 μ l 10M NH
4AC, ice bath 10min; The centrifugal 10min of 13000rpm gets supernatant; The dehydrated alcohol that adds 2 times of volumes is placed 30min for-20 ℃; The 13000 centrifugal 10min of rpm abandon supernatant; With 70% washing with alcohol 2 times; Dry, add water dissolution, in-20 ℃ of preservations.
1.2 gene clone
Adopt and merge the PCR method clone gene, corresponding Taq enzyme is available from TaKaRa company.Inquiry has obtained the Hac1 nucleic acid sequence SEQ ID NO:1 of pichia spp on GenBank.This experimental design 3 PCR primers, called after F-Hac1-EcoR1 respectively, R-Hac1-Not1 and the concrete sequence of R-Hac1-N(primer see Table 1), expand the DNA sequence dna of the Hac1 adult form mRNA of intron.
The concrete sequence of table 1 primer
First round PCR reaction: the genome that extracts in the embodiment 1.1 is as template, utilizes primers F-Hac1-EcoR1/ R-Hac1-N amplification intron upstream fragment, and the PCR condition is 95 ℃ of 4min; 94 ℃ of 40S; 55 ℃ of 30S, 30 circulations of 72 ℃ of 1.0min; 72 ℃ of 7min.Then gel reclaims the PCR product.
Second takes turns the PCR reaction: first round PCR product is template, utilizes primers F-Hac1-EcoR1/ R-Hac1-Not1 amplification to remove and contains son fusion fragment, and the PCR condition is 95 ℃ of 4min; 94 ℃ of 40S; 55 ℃ of 30S, 30 circulations of 72 ℃ of 1.0min; 72 ℃ of 7min.Then gel reclaims the PCR product.
At last, take turns the PCR product cloning to the T carrier with second; Positive colony is delivered to the large genome company of Beijing China carry out sequencing analysis.The result demonstration, the pcr amplification result is errorless, and the nucleotides sequence of its product is classified SEQ ID NO:2 as, and the aminoacid sequence of the albumen Hac1 of the nucleotide coding of SEQ ID NO:2 is SEQ ID NO:3.A plurality of clones' sequencing result is also all consistent.
The structure of embodiment 2 Pichia yeast engineerings
2.1 expression vector establishment
After in embodiment 1.2, the amplified production gel reclaims, first carry out EcoR I and Not I double digestion.Equally, Pichia anomala expression plasmid pGAPZ is also carried out EcoR I and Not I double digestion.Be the double digestion product that clone gene is connected a ℃ connection and is spent the night with expression vector with the T4 ligase enzyme.At last, import escherichia coli DH5a connecting product, obtain corresponding expression vectors called after pGAP-Hac1, the expression vector collection of illustrative plates as shown in Figure 1.Order-checking shows that the carrier of acquisition has inserted correct fragment.
2.2 competent preparation
Its deposit number of Pichia yeast engineering Pichia pastoris KDN-1(CCTCC M 209130 that can Expressing Recombinant Phytase, see that application number is 200910017741X, publication number is 101955957A, open date 2011-01-26), this experiment is take Pichia pastoris KDN-1 as transforming the host.At first picking Pichia pastoris KDN-1 30 ℃ of shaking culture in the YPD substratum are spent the night, then transfer in 100 mL YPD substratum, are cultured to OD600=1.5, ice bath 10min, and 4 ℃, the 5000 centrifugal 3min of rpm collect thalline.Aseptic double-distilled water with precooling washs thalline 2 times, then is resuspended in the electrophoretic buffer of 1 mL precooling, and this damping fluid contains 1mM MgCl
2, 10mM HEPES, 250mM sucrose, pH 7.8.
Method of the present invention can also be applied to other Pichia yeast engineering that can express source of phytase, and is not limited only to the concrete bacterial strain in embodiment.
2.3 transform and checking
Add 5 μ L linearizing recombinant plasmid pGAP-Hac1 in 80 μ L competent cells.Transform at the EMC830 electroporation with 1 mm electric shock cup, its electric shock condition is 300V, 16ms; Coat at last and contain that 100 μ g/ml bleomycin PDA are dull and stereotyped to be cultivated, select recombinant bacterial strain.
Extracting the transformant genomic dna according to the method in embodiment 1.1 is template, utilizes primer amplification purpose checking transformant.Utilize primers F in embodiment 1-Hac1-EcoR1/ R-Hac1-Not to carry out the pcr amplification goal gene.The pcr amplification condition is 94 ℃ of 3min; 94 ℃ of 30S; 58 ℃ of 30S, 30 circulations of 72 ℃ of 90S; 72 ℃ of 7min.Utilize gel to reclaim test kit and reclaim pcr amplification product and carry out sequencing analysis, through PCR reaction seed selection and checking positive transformant.
The fermentation analysis of embodiment 3 transformants
3.1 engineering bacteria shake flask fermentation
Choose at random the positive engineering strain (called after H1, H2, H3 and H4 respectively) that obtains in 4 strain embodiment 2.3 and the bacterium Pichia pastoris KDN-1 that sets out and be inoculated in respectively GM fermention medium (peptone 2%; Yeast powder extract 1%; YNB 1.34%; Phosphoric acid buffer 0.01M pH 6.0; Sodium Glutamate 1%), 4 bottles of each inoculation are namely done 4 repetitions, and then 28 ℃, the 200rpm shaking table was cultivated 72 hours, and the centrifuging and taking supernatant liquor carries out the enzymatic determination analysis.
3.2 enzyme activity determination
(1) reagent and solution
A. test solution used:
0.25mol/L acetate buffer, the 7.5mmol/L sodium phytate solution, salpeter solution (nitric acid: water=1:2),
The 100g/L ammonium molybdate solution, the 2.35g/L Ammonium Vanadate Solution
B. enzyme digestion reaction stops and nitrite ion:
Pipette 2 parts of salpeter solutions (5.4), 1 part of ammonium molybdate solution (5.5), 1 part of Ammonium Vanadate Solution (5.6) mixes use, matching while using.
C. potassium primary phosphate (KH2PO4) typical curve:
Accurately take 0.6804g at 105 ℃ of benchmark potassium primary phosphates (5.9) that dry to constant weight in the 100ml volumetric flask, (dissolving and is settled to 100ml, and concentration is 50.0mmol/L with acetate buffer.Ratio in table 1 is diluted to different concns with acetate buffer, reaction assay together with sample to be tested.Take inorganic phosphorus concentration as X-coordinate, light absorption value is ordinate zou, lists linear regression equation (y=ax+b).
Table 1 standard Dilution ratio
| The standardized solution sequence number | Amount of dilution/mL | Concentration/(μ mol/L) |
| 1 | 0.5?→?1 | 25.000 |
| 2 | 0.5?→?2 | 12.500 |
| 3 | 0.5?→?4 | 6.2500 |
| 4 | 0.5?→?8 | 3.1250 |
| 5 | 0.5?→?16 | 1.5625 |
D. measuring method
Application of sample step and process see Table 2, and reacted sample is standing 10min in water-bath, and with the centrifugal 10min of 4000r/min, supernatant liquor is with the blank zeroing of typical curve, at the blank (A of spectrophotometer 415nm wavelength place's working sample on whizzer
0) and the light absorption value of sample solution (A), A-A
0Be the actual measurement light absorption value.Calculate the activity of phytase with linear regression equation.
Table 2 reactions steps and reagent, solution usage
Phytase activity is calculated as follows
U=F×C?/（m×30）
In formula:
The activity of phytase in U-sample, U/g
The enzymic activity of C-calculated by linear regression equation according to the light absorption value of actual sample liquid, U.
Total extension rate before F-sample solution reaction.
M-sample mass, g.
30-reaction times, min.
Measuring according to the method described above the enzyme of the fermentation supernatant that obtains in embodiment 3.1 lives, result shows: the average enzyme of bacterial strain H1 is lived and is 432U/ml, the average enzyme of bacterial strain H2 is lived and is 440U/ml, and it is 420U/ml that the average enzyme of bacterial strain H3 is lived, and the average enzyme of bacterial strain H4 is lived and is 418U/ml; And the average enzyme work of starting strain is 368 U/ml.The above results shows: translational control factor Hac1 can significantly promote the expression of phytase in pichia spp.
With positive engineering strain H1 called after pichia spp SDLH-1(Pichia pastoris SDLH-1), in on December 5th, 2012 be preserved in be positioned at Lopa Nationality an ancient woman's ornament mountain, Wuhan Wuhan University " Chinese Typical Representative culture collection " center ", preserving number are CCTCC NO:M 2012503.
Claims (7)
1. recombinant expression vector, described carrier is for being used for expressing the carrier for expression of eukaryon of translational control factor Hac1 gene.
2. recombinant expression vector as claimed in claim 1, is characterized in that the nucleotides sequence of described translational control factor Hac1 gene is classified SEQ ID NO:2 as.
3. recombinant expression vector as claimed in claim 1 or 2, the aminoacid sequence that it is characterized in that described translational control factor Hac1 gene is SEQ ID NO:3.
4. recombinant expression vector as claimed in claim 1, is characterized in that described carrier for expression of eukaryon is Pichia anomala expression plasmid pGAPZ.
5. one kind is improved the method that phytase is expressed in Pichia yeast engineering, is that recombinant expression vector claimed in claim 1 is changed in the Pichia yeast engineering of Expressing Recombinant Phytase, builds new recombinant bacterial strain.
6. an engineering bacteria, be to build with method claimed in claim 5.
7. engineering bacteria as claimed in claim 6, its deposit number is CCTCC NO:M 2012503.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083373A (en) * | 2017-06-01 | 2017-08-22 | 江苏师范大学 | The recombinant yeast pichia pastoris of one plant of heterologous high efficient expression lipase and its application |
| CN113528565A (en) * | 2021-05-06 | 2021-10-22 | 广东溢多利生物科技股份有限公司 | Molecular chaperone expression vector and strain for improving secretion expression of phytase in pichia pastoris |
| CN115725635A (en) * | 2022-07-28 | 2023-03-03 | 青岛蔚蓝生物集团有限公司 | Pichia pastoris mutant strain and application thereof in production of neutral phytase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101955957A (en) * | 2009-08-21 | 2011-01-26 | 青岛康地恩生物科技有限公司 | New phytase gene and expression vector thereof |
| CN102459609A (en) * | 2009-05-20 | 2012-05-16 | 维也纳南城高等专业学院 | Eukaryotic host cell comprising an expression enhancer |
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- 2012-12-17 CN CN2012105500264A patent/CN103088052A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102459609A (en) * | 2009-05-20 | 2012-05-16 | 维也纳南城高等专业学院 | Eukaryotic host cell comprising an expression enhancer |
| CN101955957A (en) * | 2009-08-21 | 2011-01-26 | 青岛康地恩生物科技有限公司 | New phytase gene and expression vector thereof |
Non-Patent Citations (1)
| Title |
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| MOUNA GUERFAL ET AL.: "The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins", 《MICROBIAL CELL FACTORIES》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083373A (en) * | 2017-06-01 | 2017-08-22 | 江苏师范大学 | The recombinant yeast pichia pastoris of one plant of heterologous high efficient expression lipase and its application |
| CN107083373B (en) * | 2017-06-01 | 2020-07-07 | 江苏师范大学 | Recombinant pichia pastoris for heterologous high-efficiency expression of lipase and application thereof |
| CN113528565A (en) * | 2021-05-06 | 2021-10-22 | 广东溢多利生物科技股份有限公司 | Molecular chaperone expression vector and strain for improving secretion expression of phytase in pichia pastoris |
| CN113528565B (en) * | 2021-05-06 | 2023-09-29 | 广东溢多利生物科技股份有限公司 | Molecular chaperone expression vector and strain for improving secretory expression of phytase in pichia pastoris |
| CN115725635A (en) * | 2022-07-28 | 2023-03-03 | 青岛蔚蓝生物集团有限公司 | Pichia pastoris mutant strain and application thereof in production of neutral phytase |
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Application publication date: 20130508 |