CN105420269A - Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris - Google Patents
Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris Download PDFInfo
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Abstract
The invention relates to a construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris, in particular to cellulase protein gene codon optimization and construction of a cellulase protein and vitreoscilla hemoglobin protein (VHb)-co-expressed pichia pastoris system. According to the construction method, codon bias optimization is performed on the nucleotide sequence of EG II (GenBank Accession No.DQ178347.1) through Gene Designer (DNA2.0, Menlo Park, CA, USA) software, a pPIC9K-eg2 expression vector is constructed, and a recombined pichia pastoris strain is obtained by taking Pichiapastoris GS115 as a host through electrotransformation. In addition, the nucleotide sequence of the VHb (GenBank Accession No.M30794.1) is obtained from NCBI and artificially synthesized into a gene, then a pPICZalphaA-vgb expression vector is constructed, and the co-expressed pichia pastoris strain is obtained by taking the recombined pichia pastoris strain containing the EG II gene as a host through electrotransformation. Detection shows that the co-expressed strain is improved on the aspects of bacterial concentration growth and enzyme activity, wherein the OD600 value is increased by 7.2%, and the enzyme activity is improved by 2.2%.
Description
Technical field
The present invention relates to the structure of the recombinant yeast pichia pastoris system of a kind of coexpression Vitreoscilla hemoglobin gene (vgb) and cellulase protein, belong to technical field of bioengineering.
Background technology
Cellulase is the general name of a class polycomponent enzyme, by being difficult to for the benefit of bioavailable glucose of bioavailable cellulose hydrolysis, can therefore be considered to one of zymin had a high potential.Cellulase belongs to glycoside hydrolase, the cellulase system of degraded cellulose can at least comprise three class components, be respectively 1,4-BETA-D-glucancellobio-hydrolase (CBH), endoglucanase (EG) and beta-glucosidase (BG).Wherein EG acts on Mierocrystalline cellulose noncrystalline domain, hydrolysis β-Isosorbide-5-Nitrae glycosidic link, linear fibre element polymer can be blocked and generate a large amount of Mierocrystalline cellulose small molecules; CBH is hydrolyzed Isosorbide-5-Nitrae-β-D-glycosidic link, acts on linear fibre element polymer end, generates cellobiose molecule; Cellobiose hydrolysis is then become glucose by BG.Mutually acted synergistically by cellulase prozyme component, by biopolymer-Mierocrystalline cellulose the abundantest on the earth, the glucose that microorganism can utilize easily can be converted into, produce biofuel, in order to alleviate current energy shortage problem by fermentation.
Cellulase wide material sources, the biology producing cellulase comprises insect, mollusk, protozoon, bacterium and fungi.But, these self-produced enzymes innately have certain proportioning defect, with regard to current industrial application the most widely cellulase-producing fungi Trichodermareesei (Trichodermareesei), lack excision enzyme CBHII and beta-glucosidase BG in its cellulase system, cause its cellulase system enzymolysis efficiency low.In addition, no matter derive from biological self-produced enzyme system or the commercial fibres element enzyme system of manually making, wherein contained albumen reaches more than 80 and plants, even more, causes core enzyme ratio of component enzyme to be lived and reduces, and this is equally also the low reason of cellulase system enzymolysis efficiency.What is more important, in different ligno-cellulosic materials, there were significant differences for the ratio of Mierocrystalline cellulose, hemicellulose and xylogen, causes same class commercial fibres element zymin or self-produced enzyme can not play best hydrolysis result.So, optimize customization cellulase, make each component reach optimum proportioning, optimum hydrolysis effect could be obtained when minimum enzyme dosage.Therefore, in order to optimize customization cellulase, obtain cellulase list enzyme component very crucial.
In the last few years, heterogenous expression cellulose enzyme gene obtained single enzyme component is study hotspot.Pichiapastoris can realize posttranslational modification effect, as glycosylation, disulfide formation, albumen can correctly be folded, and obtain activated target protein, in addition, pichia spp does not produce endogenous lignocellulolyticenzymes component, and extracellular protein composition uncomplicated, recombinant yeast pichia pastoris bacterium fermented supernatant fluid even directly can use as single zymin without purifying, therefore, using Pichiapastoris as first-selected Host Strains, realize cellulose components heterogenous expression to obtain high density and highly purified cellulase protein component, become study hotspot.
Data shows, Vitreoscilla hemoglobin (VHb) originates from strict aerobic bacteria Vitreoscilla, and VHb is the automatic first aid albumen of one when Vitreoscilla is in anoxic condition.Hypothesis about its function shows that VHb can catch oxygen in low-oxygen environment, supplies the oxydase relevant to respiration, thus improves the effciency of respiratory and promote the generation of ATP.Cellulase protein component heterogenous expression is realized using pichia spp as Host Strains, its industrialized prerequisite must have high-quality potentiality in the high-density culture of oxygen supply deficiency, by realizing the coexpression of VHb and cellulase protein in pichia spp, product enzyme level and the industrial applications potentiality of recombinant yeast pichia pastoris can be improved.
Summary of the invention:
The object of the invention is to the pichia spp building a kind of coexpression Vitreoscilla hemoglobin gene and cellulase protein gene.
The Bichi yeast system concrete steps of coexpression VHb albumen provided by the invention and endoglucanase II are as follows:
1, EGII codon bias is optimized:
Present invention achieves the optimization of EGII codon bias, its original password subsequence is as follows:
SEQIDNO.1
Codon optimized front EGII aminoacid sequence is as shown in SEQIDNO.2.
SEQIDNO.2
The present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) to realize optimizing the EGII nucleotide sequence codon bias shown in SEQIDNO.1, and after optimizing, nucleotide sequence is as shown in SEQIDNO.3.
SEQIDNO.3
After codon is optimized, EGII aminoacid sequence is identical with SEQIDNO.2.
2, construction of expression vector pPIC9K-eg2:
Using plasmid pPIC9K as carrier, the EGII gene after optimizing is inserted promotor AOX1 downstream, 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPIC9K-eg2 expression vector.
3, recombinant bacterial strain is obtained:
Using SacI as pPIC9K-eg2 expression vector linearisation sites, realize expression vector linearizing, proceed to pichia spp by electricity.
4, vgb gene is obtained:
From NCBI, obtain Vitreoscilla hemoglobin nucleotide sequence (GenBankAccessionNo.M30794.1), add EcoRI restriction enzyme site at 5 ' end, 3 ' end adds NotI restriction enzyme site, obtains goal gene by synthetic.
5, construction of expression vector pPICZ α A-vgb:
Using plasmid pPICZ α A as carrier, vgb gene is inserted promotor AOX1 downstream, 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPICZ α A-vgb expression vector.
6, coexpression recombinant bacterial strain is obtained:
Using SacI as pPICZ α A-vgb expression vector linearisation sites, realize expression vector linearizing, proceed to the recombinant yeast pichia pastoris containing eg2 gene by electricity.
7, VHb protein-active of recombinating detects:
Utilize CO differential spectra detection method to detect recombinant yeast pichia pastoris and whether secrete activated VHb albumen.
8, enzymatic production and product enzyme level compare:
Produce enzyme by shake flask fermentation, compare the product enzyme level of EGII recombinant bacterial strain and EGII and VHb coexpression bacterial strain.
Beneficial effect of the present invention:
1, present invention achieves the optimization of Trichodermareesei endoglucanase II (EGII) gene codon skewed popularity, deflection object is Pichia yeast, reduce GC content in optimizing process, be reduced to 46.2% by 55.9%, improve recombinant yeast pichia pastoris bacterium expression efficiency.
2, the present invention is in Trichodermareesei endoglucanase II (EGII) gene codon skewed popularity optimizing process, utilize RNA secondary structure folding software RNAstructure to realize secondary structure to the nucleotide sequence after optimization to fold, and adjust base sequence, initiator codon end base is made to be open loop structure, be beneficial to rrna to combine, improve recombinant yeast pichia pastoris bacterium expression efficiency.
3, present invention achieves cellulase protein and Vitreoscilla hemoglobin (VHb) coexpression in pichia spp, improve the product enzyme efficiency of recombinant yeast pichia pastoris bacterium in hypoxemia process of high-density fermentation, promote the industrial production potentiality of recombinant yeast pichia pastoris bacterium.
Accompanying drawing explanation
Fig. 1: experimental considerations and scheme;
Fig. 2: eg2 nucleotide sequence contrasts before and after optimizing;
Fig. 3: eg2 aminoacid sequence contrasts before and after optimizing;
Fig. 4: pPIC9K-eg2 expression vector collection of illustrative plates;
Fig. 5: 1% nucleic acid gel electrophoresis:
1:SacI single endonuclease digestion Expression vector pPIC9K-eg2,
2:EcoRI and NotI double digestion Expression vector pPIC9K-eg2;
Fig. 6: high yield restructuring EGII albumen bacterial strain screening;
Fig. 7: pPICZ α A-vgb expression vector collection of illustrative plates;
Fig. 8: 1% nucleic acid gel electrophoresis:
1:SacI single endonuclease digestion expression vector pPICZ α A-vgb,
2:EcoRI and NotI double digestion expression vector pPICZ α A-vgb;
PPICZ α A-vgb successfully constructs, and enzyme cuts rear nucleic acid electrophoresis figure
Fig. 9: high yield restructuring EGII albumen coexpression bacterial strain screening
Figure 10: bacterium colony PCR checking coexpression bacterial strain:
P: the former bacterium of pichia spp,
E:EGII recombinant yeast pichia pastoris bacterium,
VE1, VE25, VE31 etc.: EGII and VHb coexpression bacterial strain;
EGII and VHb coexpression strain fermentation supernatant liquor after Figure 11: full wavelength scanner CO gas processing;
Figure 12: EGII recombinant bacterial strain compares with enzymatic productivity with EGII and VHb coexpression strain growth:
A: strain enzyme-producing ability compares,
B: strain growth ability compares.
Embodiment
The present invention constructs the pichia spp of a kind of coexpression Vitreoscilla hemoglobin gene and Trichodermareesei endoglucanase II gene, and to improve recombinant bacterial strain oxygen utilization efficiency, thus improve the dense and enzymatic productivity of its bacterium, Constructed wetlands as shown in Figure 1.
Embodiment 1: Trichodermareesei endoglucanase II (EGII) gene codon optimization and expression vector establishment.
1, the present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) realize EGII (GenBankAccessionNo.DQ178347.1) nucleotide sequence codon bias as shown in SEQIDNO.1 to optimize, after optimizing, nucleotide sequence is as shown in SEQIDNO.3.Consistent with original amino acid by the aminoacid sequence after codon optimized, as shown in SEQIDNO.2.Using PichiapastorisGS115 as host, contrasted by nucleotide sequence after codon bias optimization and original nucleotide sequences and see Fig. 2, contrasted by aminoacid sequence after codon bias optimization and original amino acid and see Fig. 3.
2, with 5 ' end restriction enzyme site for EcoRI, 3 ' end restriction enzyme site be NotI as double enzyme site, insert pPIC9K plasmid vector AOX1 promotor downstream, construction of expression vector pPIC9K-eg2, Fig. 4 is shown in by expression vector collection of illustrative plates.And by double digestion, expression vector is verified, to determine that Expression vector pPIC9K-eg2 successfully constructs, enzyme is cut rear nucleic acid electrophoresis figure and is seen Fig. 5.
Embodiment 2: linearizing expression vector also carries out electricity conversion and screening superior strain.
1, using SacI as pPIC9K-eg2 expression vector linearisation sites, realize expression vector linearizing, nucleic acid electrophoresis is verified the plasmid after linearizing, sees Fig. 5.PichiapastorisGS115 is proceeded to by electricity, obtain recombinant bacterial strain, Geneticin G-418 concentration gradient is utilized to screen multiple copied transformant, superior strain can be obtained by shake flask fermentation by the bacterium colony of normal growth in high density resistant panel, utilize in 120h place DNS method to measure fermented supernatant fluid CMC enzyme to live, see Fig. 6, wherein, 1 unit enzyme unit definition alive is under 50 DEG C of water bath condition, can transform the enzyme amount of 1 μm of ol substrate in 1 minute.
Embodiment 3: the acquisition of Vitreoscilla hemoglobin (VHb) gene and expression vector establishment.
1, from NCBI, obtain Vitreoscilla hemoglobin gene (GenBankAccessionNo.M30794.1), add EcoRI restriction enzyme site at 5 ' end, 3 ' end adds NotI restriction enzyme site, obtains goal gene by synthetic.
2, with 5 ' end restriction enzyme site for EcoRI, 3 ' end restriction enzyme site be NotI as double enzyme site, insert pPICZ α A plasmid vector AOX1 promotor downstream, construction of expression vector pPICZ α A-vgb, Fig. 7 is shown in by expression vector collection of illustrative plates.And by double digestion, expression vector is verified, to determine that expression vector pPICZ α A-vgb successfully constructs, enzyme is cut rear nucleic acid electrophoresis figure and is seen Fig. 8.
Embodiment 4: linearizing expression vector pPICZ α A-vgb also carries out electricity conversion and screening coexpression superior strain.
1, using SacI as pPICZ α A-vgb expression vector linearisation sites, realize expression vector linearizing, nucleic acid electrophoresis is verified the plasmid after linearizing, sees Fig. 8.The restructuring PichiapastorisGS115 containing eg2 gene is proceeded to by electricity, obtain coexpression recombinant bacterial strain, microbiotic Zeocin concentration gradient is utilized to screen multiple copied transformant, superior strain can be obtained by shake flask fermentation by the bacterium colony of normal growth in high density resistant panel, sample in 72h and utilize DNS method to measure fermented supernatant fluid CMC enzyme and live, see Fig. 9.In addition, carry out bacterium colony PCR to these bacterial strains, in order to verify that vgb gene and eg2 gene are present in pichia spp host chromosome jointly, result as shown in Figure 10.
Embodiment 5: restructuring VHb protein-active detects.
Can be there is irreversible combination in CO and VHb albumen, form stable porphyrin ring structure, and this structure has absorption peak at a particular wavelength, and this method therefore can be utilized to detect restructuring VHb albumen.
EGII recombinant bacterial strain and EGII and VHb coexpression bacterial strain are accessed fermention medium respectively, after fermentation culture 120h, gets fermented liquid supernatant, use excessive Na
2s
2o
4cO, as after reductive agent process fermented liquid 10min, is passed into 3min in fermented liquid by (vat powder).Using EGII recombinant bacterial strain fermented supernatant fluid as blank, Hitachi U3900 spectrophotometer is utilized to carry out full wavelength scanner, wavelength region 300 to 500nm.Result as shown in figure 11.
Embodiment 6:EGII recombinant bacterial strain compares with the product enzyme level of EGII and VHb coexpression bacterial strain.
1, using BMMY as culture medium, using methyl alcohol as inductor, realize producing enzyme with methyl alcohol addition 1.5%, get 72h, 96h and 120h sample and carry out enzyme activity determination and the dense mensuration of bacterium, compare from biomass and enzyme activity aspect EGII recombinant bacterial strain and EGII and VHb coexpression bacterial strain, result as shown in figure 12.
Claims (9)
1. the construction process of coexpression oxyphorase VHb and cellulase protein in pichia spp, is characterized in that following steps:
1) codon bias optimization: the present invention utilizes software GeneDesigner to realize the EGII nucleotide sequence codon bias optimization shown in SEQIDNO.1, and after optimizing, nucleotide sequence is as shown in SEQIDNO.3.
2) pPIC9K-eg2 expression vector establishment: take plasmid pPIC9K as carrier, inserts promotor AOX1 downstream by the EGII gene after optimizing, and 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPIC9K-eg2 expression vector.
3) recombinant bacterial strain is obtained: using SacI as pPIC9K-eg2 expression vector linearisation sites, realize expression vector linearizing, proceed to pichia spp by electricity.
4) obtain vgb gene: from NCBI, obtain Vitreoscilla hemoglobin gene, add EcoRI restriction enzyme site at 5 ' end, 3 ' end adds NotI restriction enzyme site, obtains goal gene by synthetic.
5) pPICZ α A-vgb expression vector establishment: using plasmid pPICZ α A as carrier, inserts promotor AOX1 downstream by vgb gene, and 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPICZ α A-vgb expression vector.
6) obtain height by high density Geneticin G-418 and the Double screening of bleomycin and copy coexpression bacterial strain.
7) the product enzyme level of EGII recombinant bacterial strain and EGII and VHb coexpression bacterial strain is compared.
2. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 1) codon bias optimization, codon mainly refers to coding endo cellulase, exocellulase, β-grape and Glycosylase, and zytase and xylosidase, also relate to hemicellulose side-chain hydrolysis involved enzyme class in addition.
3. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its coexpression recombinant bacterial strain described, refers to recombinant yeast pichia pastoris bacterium.
4. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its recombinant bacterial strain can express cellulase protein by stability and high efficiency, and coexpression Pichi strain can produce activated VHb albumen while cellulase protein.
5. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 1) EGII be by the Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II gene such as shown in SEQIDNO.1 or SEQIDNO.3 coded by nucleotide sequence.
6. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 1) in the EGII gene codon skewed popularity that describes optimize, after optimizing, EGII nucleotide sequence is as shown in SEQIDNO.3.
7. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 1) in the EGII gene that describes, its aminoacid sequence is as shown in SEQIDNO.2.
8. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 2) in the expression vector that describes, be pPIC9K-eg2 expression vector.
9. the construction process of coexpression oxyphorase VHb and cellulase protein in a kind of pichia spp according to claim 1, its step 5) in the expression vector that describes, be pPIC9K-vgb expression vector.
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| CN108410842A (en) * | 2017-02-10 | 2018-08-17 | 中国科学院微生物研究所 | A kind of recombinant bacterium and its application in producing cellulase |
| CN109652437A (en) * | 2019-02-21 | 2019-04-19 | 中国农业大学 | It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application |
| CN109971784A (en) * | 2018-09-26 | 2019-07-05 | 天津科技大学 | Heterogenous expression endoglucanase EG II in a kind of Pichia pastoris, the construction method of EG IV, EG V |
| CN110343624A (en) * | 2018-04-03 | 2019-10-18 | 中国科学院微生物研究所 | A kind of recombinant bacterium and its application in raising yield of cellulase |
| CN110699269A (en) * | 2019-10-16 | 2020-01-17 | 东南大学 | Method for maintaining and culturing pichia pastoris transformant by using double antibiotics |
| CN110863005A (en) * | 2019-12-30 | 2020-03-06 | 河南省科学院生物研究所有限责任公司 | Method for improving α -galactosidase constitutive expression quantity in pichia pastoris engineering bacteria through co-expression of hemoglobin |
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| CN108410842A (en) * | 2017-02-10 | 2018-08-17 | 中国科学院微生物研究所 | A kind of recombinant bacterium and its application in producing cellulase |
| CN110343624B (en) * | 2018-04-03 | 2021-06-15 | 中国科学院微生物研究所 | Recombinant strain and application thereof in improving yield of cellulase |
| CN110343624A (en) * | 2018-04-03 | 2019-10-18 | 中国科学院微生物研究所 | A kind of recombinant bacterium and its application in raising yield of cellulase |
| CN109971784A (en) * | 2018-09-26 | 2019-07-05 | 天津科技大学 | Heterogenous expression endoglucanase EG II in a kind of Pichia pastoris, the construction method of EG IV, EG V |
| CN109652437A (en) * | 2019-02-21 | 2019-04-19 | 中国农业大学 | It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application |
| CN109652437B (en) * | 2019-02-21 | 2021-12-07 | 中国农业大学 | Construction method and application of recombinant bacterium for producing chitosanase |
| CN110699269A (en) * | 2019-10-16 | 2020-01-17 | 东南大学 | Method for maintaining and culturing pichia pastoris transformant by using double antibiotics |
| CN110951768A (en) * | 2019-12-30 | 2020-04-03 | 河南省科学院生物研究所有限责任公司 | Method for improving α -galactosidase methanol induction expression quantity in pichia pastoris engineering bacteria through co-expression of hemoglobin |
| CN110863005A (en) * | 2019-12-30 | 2020-03-06 | 河南省科学院生物研究所有限责任公司 | Method for improving α -galactosidase constitutive expression quantity in pichia pastoris engineering bacteria through co-expression of hemoglobin |
| CN112300951A (en) * | 2020-10-10 | 2021-02-02 | 广东省微生物研究所（广东省微生物分析检测中心） | Recombinant pichia pastoris and application thereof in adsorption of heavy metal ions |
| CN112300951B (en) * | 2020-10-10 | 2022-04-15 | 广东省微生物研究所（广东省微生物分析检测中心） | Recombinant pichia pastoris and application thereof in adsorption of heavy metal ions |
| CN113322270A (en) * | 2021-03-12 | 2021-08-31 | 上海国龙生物科技有限公司 | Preparation method and application of pichia pastoris for expressing mixed enzyme preparation |
| CN113549644A (en) * | 2021-05-14 | 2021-10-26 | 中南民族大学 | Recombinant yeast displaying three NSP enzymes together and construction method and application thereof |
| CN113549644B (en) * | 2021-05-14 | 2024-02-02 | 中南民族大学 | Recombinant yeast displaying three NSP enzymes together, construction method and application thereof |
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