CN115247136A - Method for preparing pichia pastoris with surface display of fructosyl transferase - Google Patents
Method for preparing pichia pastoris with surface display of fructosyl transferase Download PDFInfo
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- CN115247136A CN115247136A CN202110473167.XA CN202110473167A CN115247136A CN 115247136 A CN115247136 A CN 115247136A CN 202110473167 A CN202110473167 A CN 202110473167A CN 115247136 A CN115247136 A CN 115247136A
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- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 55
- 108010042889 Inulosucrase Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000004927 fusion Effects 0.000 claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000013604 expression vector Substances 0.000 claims abstract description 16
- 230000001131 transforming effect Effects 0.000 claims abstract description 10
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 9
- 238000003259 recombinant expression Methods 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims abstract description 6
- 230000006698 induction Effects 0.000 claims abstract description 6
- 108010075254 C-Peptide Proteins 0.000 claims description 5
- 241000235648 Pichia Species 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 238000004873 anchoring Methods 0.000 abstract description 8
- 230000002194 synthesizing effect Effects 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 description 14
- 229940107187 fructooligosaccharide Drugs 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 12
- 239000013598 vector Substances 0.000 description 10
- 229930006000 Sucrose Natural products 0.000 description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 9
- 239000005720 sucrose Substances 0.000 description 9
- 241001052560 Thallis Species 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000029087 digestion Effects 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
Abstract
The invention relates to the technical field of pichia pastoris, in particular to a method for preparing pichia pastoris with surface display of fructosyl transferase, which comprises the following steps of 1: synthesizing a fusion sequence of the improved fructosyltransferase gene and the pichia pastoris anchoring protein gene, wherein the sequence of the fused gene plus a carrier homologous arm is shown as SEQ ID No.4 or SEQ ID No. 7; step 2: constructing a pichia pastoris expression vector, and connecting or recombining the fusion sequence with the enzyme digestion linearized pichia pastoris expression vector to obtain a recombinant expression vector; and 3, step 3: transforming pichia pastoris, and transforming the constructed recombinant expression vector into pichia pastoris competent cells; and 4, step 4: screening multicopy positive clones, and screening out positive pichia pastoris containing multicopy fusion genes through high resistance; and 5: and (3) obtaining the pichia pastoris with the surface display of the fructosyl transferase after methanol induction expression. The pichia pastoris can be recycled, and the preparation cost is low.
Description
Technical Field
The invention relates to the technical field of pichia pastoris, in particular to a method for preparing pichia pastoris with a surface displaying fructosyl transferase.
Background
The existing production method of fructo-oligosaccharide (FOS) is realized by catalyzing fructosyl transfer reaction between sucrose molecules by purified fructosyltransferase. Firstly, sucrose is decomposed by fructosyl transferase to generate fructosyl or glucose, then part of the fructosyl reacts with acceptor sucrose to generate fructo-oligosaccharide, and the fructosyl transferase from microorganisms has higher enzyme activity than that of the fructosyl transferase from plants, so that the production of the fructosyl transferase mainly depends on the expression of the microorganisms and then purification. When the fructo-oligosaccharide is produced by enzyme catalysis, if the fructo-oligosaccharide is intracellular enzyme, cells are required to be firstly crushed to release the intracellular enzyme, then the enzyme is separated and purified, the purified fructosyltransferase can be used for catalyzing sucrose to generate fructo-oligosaccharide, the separated and purified enzyme can also be used for catalyzing the sucrose to generate fructo-oligosaccharide after being fixed, the enzyme is inactivated after the catalysis is finished, and only the fructo-oligosaccharide of a target product is separated.
Therefore, in the prior enzyme catalysis technology, protein purification and immobilization operations are required, but enzyme separation and purification are complex steps and lose a part of enzyme, especially intracellular enzyme extraction is more complex, cells need to be broken first to release intracellular enzyme, immobilization operation needs a carrier, carrier cost and immobilization operation cost are increased, enzyme yield and activity in the enzyme immobilization process are lost, the separated and purified enzyme cannot be reused after use, and the cost is greatly increased due to the defects. In view of this, we propose a method for preparing pichia pastoris that displays fructosyl transferase on its surface.
Disclosure of Invention
The present invention is directed to a method for preparing pichia pastoris displaying fructosyl transferase on the surface, so as to solve the problems presented in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing pichia pastoris displaying fructosyl transferase on the surface, comprising the following steps:
step 1: synthesis of fusion sequences
Wherein, the fructosyltransferase gene sequence shown as SEQ ID No.1 and the pichia pastoris anchoring protein gene sequence shown as SEQ ID No.2 are fused through a connecting peptide sequence shown as SEQ ID No.3 to obtain a fusion sequence of the improved fructosyltransferase gene and the pichia pastoris anchoring protein gene, and the fused gene plus carrier homologous arm sequence is shown as SEQ ID No.4 or SEQ ID No. 7;
step 2: construction of Pichia expression vector
Connecting or recombining the fusion sequence with a digestion linearized pichia pastoris expression vector to obtain a recombinant expression vector;
and step 3: transformation of Pichia pastoris
Transforming the constructed recombinant expression vector into a pichia pastoris competent cell;
and 4, step 4: screening for multicopy positive clones
Screening out positive pichia pastoris containing multi-copy fusion genes through high resistance;
and 5: obtaining pichia pastoris displaying fructosyl transferase on surface
And (3) obtaining the pichia pastoris with the surface display of the fructosyl transferase after methanol induction expression.
Compared with the prior art, the invention has the beneficial effects that: the method for preparing the pichia pastoris with the surface display of the fructosyl transferase has perfect gene expression regulation and control, protein folding and secretion mechanisms, is an ideal eukaryotic expression host, and can be used for expressing functional proteins needing post-translational modification. The yeast is a unicellular organism, has larger cell volume and is convenient for screening and separating cells. The yeast has the characteristics of simple genetic operation, fast growth, safety and no toxicity, and is an excellent choice for producing food-grade products such as oligosaccharide. The anchoring protein can be connected to a glucan skeleton of the inner layer of the yeast cell wall through a covalent bond, so that the foreign protein expressed by fusion is firmly displayed on the surface of the yeast cell. The displayed protein can tolerate the extraction of SDS;
meanwhile, the pichia pastoris with fructosyl transferase displayed on the surface and capable of being reused can directly catalyze sucrose to generate fructo-oligosaccharide without enzyme separation, purification and immobilization operation steps, and can be reused.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for preparing Pichia pastoris with surface display of fructosyl transferase, comprising the steps of:
(1) Optimizing fructosyltransferase gene sequence as SEQ ID No.1;
(2) Optimizing pichia pastoris anchoring protein GCW21 gene sequence as SEQ ID No.2;
(3) Optimizing a Linker sequence of the connecting peptide, such as SEQ ID No.3;
(4) Chemically synthesizing a fusion sequence of the improved fructosyltransferase gene shown in SEQ ID No.4 and a pichia pastoris anchoring protein gene, wherein two ends of the fusion sequence comprise homologous arms at two sides of an EcoRI enzyme cutting site of a pichia pastoris expression vector pPIC 9K;
(5) The vector pPIC9K is subjected to single enzyme digestion by EcoRI, and the enzyme digestion system is as follows:
| plasmids | 48μL |
| EcoRI | 6μL |
| 10X buffer | 6μL |
| 60μL |
(6) Recombining the fusion sequence with the pPIC9K vector after enzyme digestion to obtain a recombinant expression vector, reacting for 1h at 50 ℃, wherein a recombinant system is as follows:
| linearized vector | 5μL |
| Fusion fragment | 10μL |
| 2X ClonExpress Mix | 25μL |
| ddH2O to | 50μL |
(7) Transforming the recombinant product into an escherichia coli Top10 competent cell;
(8) Plates coated with ampicillin;
(9) Designing a bacteria detection primer: the upstream primer PF1 is CGGCAGCATGTCTTTACTTGA, and the downstream primer PR1 is GCATCTGCATCTACCAA;
(10) Detecting positive clones by PCR;
(11) Carrying out amplification culture and extracting plasmids by using a plasmid miniextraction kit;
(12) The plasmid was linearized by digestion with SacI as follows:
| plasmids | 48μL |
| SacI | 6μL |
| 10X buffer | 6μL |
| 60μL |
(13) Running 1% agarose gel to confirm complete linearization of recombinant plasmid;
(14) Electrically transforming the linearized plasmid into a pichia competent cell, carrying out shake cultivation for 1h under the condition of 200-260rpm, and centrifuging at 5000rpm for 5min to collect thalli;
(15) Coating the thallus on His defect flat plate, culturing for 2-3 days, and selecting positive single colony;
(16) Screening pichia pastoris containing multi-copy fusion genes through a plate of 4mg/mLG 418;
(17) Transferring the pichia pastoris displaying fructosyltransferase into a 50mL centrifuge tube containing a 10mL BMGY culture base band air-permeable plug, and performing shake culture at 30 ℃ and 200-260rpm until OD600=1-7;
(18) Centrifuging at 5000rpm for 5min to collect thallus;
(19) The thalli is re-suspended in a BMM culture medium, shaking culture is carried out under the condition of 200-260rpm, 0.5 percent methanol is added every 24 hours for induction, and the culture lasts for 5-7 days;
(20) Centrifuging at 5000rpm for 5min to collect thallus and obtain Pichia pastoris P.pastoris FTase-Linker-GCW21 with surface display of fructosyl transferase;
(21) The thalli is directly used for catalyzing sucrose to produce fructo-oligosaccharide.
Example 2
A method for preparing Pichia pastoris that displays fructosyl transferase on the surface, comprising the steps of:
(1) Optimizing the sequences shown as SEQ ID No.5 and SEQ ID No. 6. The SEQ ID No.5 sequence comprises a fructosyltransferase gene sequence and a part of a Linker peptide Linker sequence. The SEQ ID No.6 sequence comprises a connecting peptide Linker sequence and an optimized anchoring protein GCW21 sequence. The left end of the SEQ ID No.5 sequence and the right end of the SEQ ID No.6 sequence comprise a vector homology arm;
(2) Chemically synthesizing sequences shown as SEQ ID No.5 and SEQ ID No. 6;
(3) Designing primers at the beginning position of the SEQ ID No.5 sequence and the end position of the SEQ ID No.6 sequence: upstream primer PF2 is GAGAGGCTGAAGCTTACGTA, downstream primer PR2 is TGTCTAAGGCGAATTAATTC;
(4) Synthesizing a fusion sequence shown as SEQ ID No.7 by using an overlapping PCR method;
(5) The vector pPIC9K and the fusion sequence shown in SEQ ID No.7 are subjected to EcoRI and Not1 double enzyme digestion, and the enzyme digestion system is as follows:
| plasmids | 42μL |
| EcoRI | 6μL |
| Not1 | 6μL |
| 10X buffer | 6μL |
| 60μL |
(6) Connecting the fused sequence after enzyme digestion with a pPIC9K vector to obtain a recombinant expression vector, wherein the connecting system is as follows:
| linearized vector | 5μL |
| Fusion fragment | 10μL |
| T4 ligase | 5μL |
| ddH2O to | 50μL |
(7) Transforming the ligation product into an escherichia coli Top10 competent cell;
(8) Plates coated with ampicillin;
(9) Designing a bacteria detection primer: an upstream primer PF1 is CGGCAGCATGTCTTTACTTGA, and a downstream primer PR1 is GCATCTGCATCTACCAA;
(10) Detecting positive clones by PCR;
(11) Carrying out amplification culture and extracting plasmids by using a plasmid miniextraction kit;
(12) The plasmid was linearized by digestion with SacI as follows:
| plasmids | 48μL |
| SacI | 6μL |
| 10X buffer | 6μL |
| 60μL |
(13) Running 1% agarose gel to confirm complete linearization of the recombinant plasmid;
(14) Electrically transforming the linearized plasmid into a pichia pastoris competent cell, carrying out shake cultivation for 1h under the condition of 200-260rpm, and centrifuging at 5000rpm for 5min to collect thalli;
(15) Coating the thalli on a His defect flat plate for culturing for 2-3 days, and selecting a positive single colony;
(16) The pichia pastoris containing the multi-copy fusion gene is screened out by a plate of G418 with the concentration of 4 mg/mL;
(17) Transferring the pichia pastoris containing the multi-copy fusion gene into a 50mL centrifuge tube containing a 10mL BMGY culture baseband vent plug, and performing shake culture at 30 ℃ and 200-260rpm until OD600=1-7;
(18) Centrifuging at 5000rpm for 5min to collect thallus;
(19) The thalli is resuspended in BMM culture medium, shake cultivation is carried out under the condition of 200-260rpm, 0.5 percent methanol is added every 24 hours for induction, and cultivation is carried out for 5-7 days;
(20) Centrifuging at 5000rpm for 5min to collect thallus and obtain Pichia pastoris P.pastoris FTase-Linker-GCW21 with surface display of fructosyl transferase;
(21) The thallus is directly used for catalyzing sucrose to produce fructo-oligosaccharide.
Example 3
A method for preparing Pichia pastoris with surface display of fructosyl transferase, comprising the steps of:
(1) And optimizing the sequence shown as SEQ ID No.7, wherein the sequence comprises a fructosyltransferase gene sequence, a connecting peptide Linker sequence and an anchoring protein GCW21 sequence. Both ends of the SEQ ID No.7 sequence contain vector homology arms;
(2) Splitting the SEQ ID No.7 sequence into a plurality of long primers with positive and negative intervals, wherein each primer is 50-70nt long;
(3) A series of forward and reverse spaced primers are synthesized as PCR templates, and adjacent forward and reverse spaced primers have an overlapping region. Designing an actual primer at the beginning position of the SEQ ID No.5 sequence and the end position of the SEQ ID No.6 sequence: upstream primer PF2 is GAGAGGCTGAAGCTTACGTA, downstream primer PR2 is TGTCTAAGGCGAATTAATTC;
(4) Synthesizing a fusion sequence shown in SEQ ID No.7 by using an overlapping PCR method;
(5) The vector pPIC9K is subjected to double digestion by EcoRI and Not1, and the digestion system is as follows:
| plasmids | 42μL |
| EcoRI | 6μL |
| Not1 | 6μL |
| 10X buffer | 6μL |
| 60μL |
(6) Recombining the vector sequence after enzyme digestion and the synthesized fusion sequence shown in SEQ ID No.7 to obtain a recombinant expression vector, wherein the recombinant system is as follows:
(7) Transforming the recombinant product into an escherichia coli Top10 competent cell;
(8) Plates coated with ampicillin;
(9) Designing a bacteria detection primer: the upstream primer PF1 is CGGCAGCATGTCTTTACTTGA, and the downstream primer PR1 is GCATCTGCATCTACCAA;
(10) Detecting positive clones by PCR;
(11) Carrying out amplification culture and extracting plasmids by using a plasmid miniextraction kit;
(12) The plasmid was linearized by digestion with SacI as follows:
| plasmids | 48μL |
| SacI | 6μL |
| 10X buffer | 6μL |
| 60μL |
(13) Running 1% agarose gel to confirm complete linearization of recombinant plasmid;
(14) Electrically transforming the linearized plasmid into a pichia pastoris competent cell, carrying out shake cultivation for 1h under the condition of 200-260rpm, and centrifuging at 5000rpm for 5min to collect thalli;
(15) Coating the thallus on His defect flat plate, culturing for 2-3 days, and selecting positive single colony;
(16) The pichia pastoris containing the multi-copy fusion gene is screened out through a plate of G418 with the concentration of 4 mg/mL;
(17) Transferring the pichia pastoris displaying fructosyltransferase into a 50mL centrifuge tube containing a 10mL BMGY culture base band air-permeable plug, and performing shake culture at 30 ℃ and 200-260rpm until OD600=1-7;
(18) Centrifuging at 5000rpm for 5min to collect thallus;
(19) The thalli is re-suspended in a BMM culture medium, shaking culture is carried out under the condition of 200-260rpm, 0.5 percent methanol is added every 24 hours for induction, and the culture lasts for 5-7 days;
(20) Centrifuging at 5000rpm for 5min to collect thallus and obtain Pichia pastoris P.pastoris FTase-Linker-GCW21 with fructosyl transferase displayed on the surface;
(21) The thallus is directly used for catalyzing sucrose to produce fructo-oligosaccharide.
The experimental results show that the method can simplify the preparation process of the fructo-oligosaccharide, and omits the complicated steps of cell disruption and enzyme separation and purification. Nor expensive enzyme immobilization equipment. In addition, the pichia pastoris with the fructosyl transferase displayed on the surface, which is provided by the method, can be recycled, so that the cost is greatly saved. Therefore, the invention can be used for the industrial preparation of fructo-oligosaccharide.
Sequence listing
SEQ ID No.1
ATGTATATCAAACCATTCATCCTACCTGCCCTTGCGGCGGTTGCGCAAGCTGCCAGCTATTC GGGAGACCTTCGGCCTCAAACTCACTCCTCTCCACCTTCCAATTTCACGAACGATCCAAGCGGTCT CTTCTATGATAGCAAGAGGGGCGTGTATCACTTATACTATCAGTATAAGCCTACAGCGACAGTCTC TGGGAATCAGCACTGGGGTCATGCCACCAGCCCTGATCTATCGCACTGGACGAATCAACGTATCGC CCTCGCTGGGGATAAGCCAGAGGAGTATATCTTCTCAGGCTCTGCTGTCGTGGACAGCAACAACAG CACAGCAATATTTCCGGAACAGGACGATGGCGTCATAGCCATCTACACAGTAAATACCCCGACACT GGAAACACATCTCATTCCTTCTTCTAGCAATGGTGGATACACTTTCACCAAGTATGAGAACAACGA GGTCATCGACATTGGCTCAAAGACGTTCCGTGACCCGGAGGTGGTGCGGCATCCTGAAACTACGCA ATGGGTGATGACACTTGCCTATGCGGAGAACTTGGTCATCCGATTTTATACCTCGCCCAATCTCAA AGACTGGTCGACTCGGACGTATATCACGCTGGAACGGCAGACGGGCGACCAATTCGAATGCCCCAA CTTGGTCAAGTTTTCGGTGACTCACGCTGAGTCTGAGGAGACGTGCATGTACGTGCTTTTCCTTTC CGTGTCCACTGGCGCTCCTCTTGGAGGTTCGCGAACACTTTATGTGGACGGCGATTTCTACGGCAG CCATTACACCTCTGAAGTTGCTCAGAAGATACTATTTGAATTCTCCAAGTACAACTATCCGGCTCA GTGCTATTCTGGTATTCCTGAGAACGAGCCTCCTGTCTCCATCGGGTGGCCTAGTACGTGCCATTA CACGGAAGAAGTCCCAACTGGCCCGCTTGAAGGCTGGAGGAGCGCCATGACTTTACCTAGAACGCA GACCATAAGCAACGTCAACGGAGTCTGGACTGTGACTCACAGCCCGTTTGAAAGCCCATCGGATCC TAAGGGCAGACAGCTTGTGAGCAAGAGTGTCCACTCTGGGGATGTGAAGACGAAGTTATCCGGGGT CCCATCGAATGCGATATATTTCAATGTGACACTGAAGGGCATCGATGTTACCAGGTCTACCGGACG CCTAAACTTCAGCTTTTCCTCCTCTGTATCGGGTGAATTCCTCGACGGAGGGGTGTCTCTGGATGA TAGTTCTCTCTGGATGACCAGAGCTGGATCCCATCTATTCACGTTTGAAGCTAGTGGAAATTATAC ATCATCTTCTACAACATCTATCACCCCCTTTGGCAACAGCATATTCACCTTCTCGGGCGTTATTGA CCGCTCTGTGTTTGAAGTCCTCATTAGTGAGGCCGGGATCCAAAGCGGAACCATGACTTTCTTCCC AAGCTCCCCACTCGATACCTTGGCTCTTACCGGAGAGGATCTCAGAGATGGGCCTAGCGTTAGTGT TAAGGCTTGGGGCCTTCAGTGCCGCAGGAATCGAACAACAGCCAGTAAGAGGTTCCGCGCC
SEQ ID No.2
ATGTCTTACTTGAAAATTTCCGCTTTCCTTTCAGTTTTGTCCGTCGCCTTGGCCGACCAACG AATCTCTGTCACCGTTGTTGGTGATGGTATCAACACAGGACTGAGATCCGGAGGGTCGCATTTTGA GGCTGGACCAAATGCTGCTGGTACCCCCTTAGATTTGATCCTGTATGAGCCGTCTGGTTTCTTGGT AGATGCAGCAGATGCTTCCAAGTACGTTGGTTGGGATGTTGCAGCTGGCACTTCTTTGACTTCTTT GCCACCAGACCAAGGAGGCAAAGATTGGGGAATCGTTGCCGGTAACCTCAGATTCAACGTTGGAGG TACTACATTCTATGCTTGTGAGACTAGGACCGGTGTTTGGGAAGTAAAGAGTTACGAAGCTAGTGG ATGCAACGCTGTGGTGCTTTCCGTAGCTAGTCACCCAGTTCCTTCTTCCAGTTCTTCCAGTTCTTC CCATGCCCCAACTTCCTCTGTTCCATCTACTTCGTCTCATGTGAGCCCAACTACCACTCAACCTCC TCACACAACCAGTTCTCACACCATCCACACATCAACTACCTTGACGACATCAGGTAGGAATGACTC GAACCACTCCAACCATACCATCCCACCAGTTCCAACCGGTGCCGCTATGGGAGTCTCTAGCAACTA TGGTTTGTTGGTTGCAGCTGGAATTGCCGCCGCTGCTTTGTTATAA
SEQ ID No.3
GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGCAGCGGCGGCGGCGGCAGC
SEQ ID No.4
GAGAGGCTGAAGCTTACGTAGAATTCATGTATATCAAACCATTCATCCTACCTGCCCTTGCG GCGGTTGCGCAAGCTGCCAGCTATTCGGGAGACCTTCGGCCTCAAACTCACTCCTCTCCACCTTCC AATTTCACGAACGATCCAAGCGGTCTCTTCTATGATAGCAAGAGGGGCGTGTATCACTTATACTAT CAGTATAAGCCTACAGCGACAGTCTCTGGGAATCAGCACTGGGGTCATGCCACCAGCCCTGATCTA TCGCACTGGACGAATCAACGTATCGCCCTCGCTGGGGATAAGCCAGAGGAGTATATCTTCTCAGGC TCTGCTGTCGTGGACAGCAACAACAGCACAGCAATATTTCCGGAACAGGACGATGGCGTCATAGCC ATCTACACAGTAAATACCCCGACACTGGAAACACATCTCATTCCTTCTTCTAGCAATGGTGGATAC ACTTTCACCAAGTATGAGAACAACGAGGTCATCGACATTGGCTCAAAGACGTTCCGTGACCCGGAG GTGGTGCGGCATCCTGAAACTACGCAATGGGTGATGACACTTGCCTATGCGGAGAACTTGGTCATC CGATTTTATACCTCGCCCAATCTCAAAGACTGGTCGACTCGGACGTATATCACGCTGGAACGGCAG ACGGGCGACCAATTCGAATGCCCCAACTTGGTCAAGTTTTCGGTGACTCACGCTGAGTCTGAGGAG ACGTGCATGTACGTGCTTTTCCTTTCCGTGTCCACTGGCGCTCCTCTTGGAGGTTCGCGAACACTT TATGTGGACGGCGATTTCTACGGCAGCCATTACACCTCTGAAGTTGCTCAGAAGATACTATTTGAA TTCTCCAAGTACAACTATCCGGCTCAGTGCTATTCTGGTATTCCTGAGAACGAGCCTCCTGTCTCC ATCGGGTGGCCTAGTACGTGCCATTACACGGAAGAAGTCCCAACTGGCCCGCTTGAAGGCTGGAGG AGCGCCATGACTTTACCTAGAACGCAGACCATAAGCAACGTCAACGGAGTCTGGACTGTGACTCAC AGCCCGTTTGAAAGCCCATCGGATCCTAAGGGCAGACAGCTTGTGAGCAAGAGTGTCCACTCTGGG GATGTGAAGACGAAGTTATCCGGGGTCCCATCGAATGCGATATATTTCAATGTGACACTGAAGGGC ATCGATGTTACCAGGTCTACCGGACGCCTAAACTTCAGCTTTTCCTCCTCTGTATCGGGTGAATTC CTCGACGGAGGGGTGTCTCTGGATGATAGTTCTCTCTGGATGACCAGAGCTGGATCCCATCTATTC ACGTTTGAAGCTAGTGGAAATTATACATCATCTTCTACAACATCTATCACCCCCTTTGGCAACAGC ATATTCACCTTCTCGGGCGTTATTGACCGCTCTGTGTTTGAAGTCCTCATTAGTGAGGCCGGGATC CAAAGCGGAACCATGACTTTCTTCCCAAGCTCCCCACTCGATACCTTGGCTCTTACCGGAGAGGAT CTCAGAGATGGGCCTAGCGTTAGTGTTAAGGCTTGGGGCCTTCAGTGCCGCAGGAATCGAACAACA GCCAGTAAGAGGTTCCGCGCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC ATGTCTTACTTGAAAATTTCCGCTTTCCTTTCAGTTTTGTCCGTCGCCTTGGCCGACCAACGAATC TCTGTCACCGTTGTTGGTGATGGTATCAACACAGGACTGAGATCCGGAGGGTCGCATTTTGAGGCT GGACCAAATGCTGCTGGTACCCCCTTAGATTTGATCCTGTATGAGCCGTCTGGTTTCTTGGTAGAT GCAGCAGATGCTTCCAAGTACGTTGGTTGGGATGTTGCAGCTGGCACTTCTTTGACTTCTTTGCCA CCAGACCAAGGAGGCAAAGATTGGGGAATCGTTGCCGGTAACCTCAGATTCAACGTTGGAGGTACT ACATTCTATGCTTGTGAGACTAGGACCGGTGTTTGGGAAGTAAAGAGTTACGAAGCTAGTGGATGC AACGCTGTGGTGCTTTCCGTAGCTAGTCACCCAGTTCCTTCTTCCAGTTCTTCCAGTTCTTCCCAT GCCCCAACTTCCTCTGTTCCATCTACTTCGTCTCATGTGAGCCCAACTACCACTCAACCTCCTCAC ACAACCAGTTCTCACACCATCCACACATCAACTACCTTGACGACATCAGGTAGGAATGACTCGAAC CACTCCAACCATACCATCCCACCAGTTCCAACCGGTGCCGCTATGGGAGTCTCTAGCAACTATGGT TTGTTGGTTGCAGCTGGAATTGCCGCCGCTGCTTTGTTATAACCTAGGGCGGCCGCGAATTA
SEQ ID No.5
GAGAGGCTGAAGCTTACGTAGAATTCATGTATATCAAACCATTCATCCTACCTGCCCTTGCG GCGGTTGCGCAAGCTGCCAGCTATTCGGGAGACCTTCGGCCTCAAACTCACTCCTCTCCACCTTCC AATTTCACGAACGATCCAAGCGGTCTCTTCTATGATAGCAAGAGGGGCGTGTATCACTTATACTAT CAGTATAAGCCTACAGCGACAGTCTCTGGGAATCAGCACTGGGGTCATGCCACCAGCCCTGATCTA TCGCACTGGACGAATCAACGTATCGCCCTCGCTGGGGATAAGCCAGAGGAGTATATCTTCTCAGGC TCTGCTGTCGTGGACAGCAACAACAGCACAGCAATATTTCCGGAACAGGACGATGGCGTCATAGCC ATCTACACAGTAAATACCCCGACACTGGAAACACATCTCATTCCTTCTTCTAGCAATGGTGGATAC ACTTTCACCAAGTATGAGAACAACGAGGTCATCGACATTGGCTCAAAGACGTTCCGTGACCCGGAG GTGGTGCGGCATCCTGAAACTACGCAATGGGTGATGACACTTGCCTATGCGGAGAACTTGGTCATC CGATTTTATACCTCGCCCAATCTCAAAGACTGGTCGACTCGGACGTATATCACGCTGGAACGGCAG ACGGGCGACCAATTCGAATGCCCCAACTTGGTCAAGTTTTCGGTGACTCACGCTGAGTCTGAGGAG ACGTGCATGTACGTGCTTTTCCTTTCCGTGTCCACTGGCGCTCCTCTTGGAGGTTCGCGAACACTT TATGTGGACGGCGATTTCTACGGCAGCCATTACACCTCTGAAGTTGCTCAGAAGATACTATTTGAA TTCTCCAAGTACAACTATCCGGCTCAGTGCTATTCTGGTATTCCTGAGAACGAGCCTCCTGTCTCC ATCGGGTGGCCTAGTACGTGCCATTACACGGAAGAAGTCCCAACTGGCCCGCTTGAAGGCTGGAGG AGCGCCATGACTTTACCTAGAACGCAGACCATAAGCAACGTCAACGGAGTCTGGACTGTGACTCAC AGCCCGTTTGAAAGCCCATCGGATCCTAAGGGCAGACAGCTTGTGAGCAAGAGTGTCCACTCTGGG GATGTGAAGACGAAGTTATCCGGGGTCCCATCGAATGCGATATATTTCAATGTGACACTGAAGGGC ATCGATGTTACCAGGTCTACCGGACGCCTAAACTTCAGCTTTTCCTCCTCTGTATCGGGTGAATTC CTCGACGGAGGGGTGTCTCTGGATGATAGTTCTCTCTGGATGACCAGAGCTGGATCCCATCTATTC ACGTTTGAAGCTAGTGGAAATTATACATCATCTTCTACAACATCTATCACCCCCTTTGGCAACAGC ATATTCACCTTCTCGGGCGTTATTGACCGCTCTGTGTTTGAAGTCCTCATTAGTGAGGCCGGGATC CAAAGCGGAACCATGACTTTCTTCCCAAGCTCCCCACTCGATACCTTGGCTCTTACCGGAGAGGAT CTCAGAGATGGGCCTAGCGTTAGTGTTAAGGCTTGGGGCCTTCAGTGCCGCAGGAATCGAACAACA GCCAGTAAGAGGTTCCGCGCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGC
SEQ ID No.6
GCGGCGGCGGCGGCAGCAGCAGCGGCGGCGGCGGCAGCATGTCTTACTTGAAAATTTCCGCT TTCCTTTCAGTTTTGTCCGTCGCCTTGGCCGACCAACGAATCTCTGTCACCGTTGTTGGTGATGGT ATCAACACAGGACTGAGATCCGGAGGGTCGCATTTTGAGGCTGGACCAAATGCTGCTGGTACCCCC TTAGATTTGATCCTGTATGAGCCGTCTGGTTTCTTGGTAGATGCAGCAGATGCTTCCAAGTACGTT GGTTGGGATGTTGCAGCTGGCACTTCTTTGACTTCTTTGCCACCAGACCAAGGAGGCAAAGATTGG GGAATCGTTGCCGGTAACCTCAGATTCAACGTTGGAGGTACTACATTCTATGCTTGTGAGACTAGG ACCGGTGTTTGGGAAGTAAAGAGTTACGAAGCTAGTGGATGCAACGCTGTGGTGCTTTCCGTAGCT AGTCACCCAGTTCCTTCTTCCAGTTCTTCCAGTTCTTCCCATGCCCCAACTTCCTCTGTTCCATCT ACTTCGTCTCATGTGAGCCCAACTACCACTCAACCTCCTCACACAACCAGTTCTCACACCATCCAC ACATCAACTACCTTGACGACATCAGGTAGGAATGACTCGAACCACTCCAACCATACCATCCCACCA GTTCCAACCGGTGCCGCTATGGGAGTCTCTAGCAACTATGGTTTGTTGGTTGCAGCTGGAATTGCC GCCGCTGCTTTGTTATAAGCGGCCGCGAATTAATTCGCCTTAGACA
SEQ ID No.7
GAGAGGCTGAAGCTTACGTAGAATTCATGTATATCAAACCATTCATCCTACCTGCCCTTGCG GCGGTTGCGCAAGCTGCCAGCTATTCGGGAGACCTTCGGCCTCAAACTCACTCCTCTCCACCTTCC AATTTCACGAACGATCCAAGCGGTCTCTTCTATGATAGCAAGAGGGGCGTGTATCACTTATACTAT CAGTATAAGCCTACAGCGACAGTCTCTGGGAATCAGCACTGGGGTCATGCCACCAGCCCTGATCTA TCGCACTGGACGAATCAACGTATCGCCCTCGCTGGGGATAAGCCAGAGGAGTATATCTTCTCAGGC TCTGCTGTCGTGGACAGCAACAACAGCACAGCAATATTTCCGGAACAGGACGATGGCGTCATAGCC ATCTACACAGTAAATACCCCGACACTGGAAACACATCTCATTCCTTCTTCTAGCAATGGTGGATAC ACTTTCACCAAGTATGAGAACAACGAGGTCATCGACATTGGCTCAAAGACGTTCCGTGACCCGGAG GTGGTGCGGCATCCTGAAACTACGCAATGGGTGATGACACTTGCCTATGCGGAGAACTTGGTCATC CGATTTTATACCTCGCCCAATCTCAAAGACTGGTCGACTCGGACGTATATCACGCTGGAACGGCAG ACGGGCGACCAATTCGAATGCCCCAACTTGGTCAAGTTTTCGGTGACTCACGCTGAGTCTGAGGAG ACGTGCATGTACGTGCTTTTCCTTTCCGTGTCCACTGGCGCTCCTCTTGGAGGTTCGCGAACACTT TATGTGGACGGCGATTTCTACGGCAGCCATTACACCTCTGAAGTTGCTCAGAAGATACTATTTGAA TTCTCCAAGTACAACTATCCGGCTCAGTGCTATTCTGGTATTCCTGAGAACGAGCCTCCTGTCTCC ATCGGGTGGCCTAGTACGTGCCATTACACGGAAGAAGTCCCAACTGGCCCGCTTGAAGGCTGGAGG AGCGCCATGACTTTACCTAGAACGCAGACCATAAGCAACGTCAACGGAGTCTGGACTGTGACTCAC AGCCCGTTTGAAAGCCCATCGGATCCTAAGGGCAGACAGCTTGTGAGCAAGAGTGTCCACTCTGGG GATGTGAAGACGAAGTTATCCGGGGTCCCATCGAATGCGATATATTTCAATGTGACACTGAAGGGC ATCGATGTTACCAGGTCTACCGGACGCCTAAACTTCAGCTTTTCCTCCTCTGTATCGGGTGAATTC CTCGACGGAGGGGTGTCTCTGGATGATAGTTCTCTCTGGATGACCAGAGCTGGATCCCATCTATTC ACGTTTGAAGCTAGTGGAAATTATACATCATCTTCTACAACATCTATCACCCCCTTTGGCAACAGC ATATTCACCTTCTCGGGCGTTATTGACCGCTCTGTGTTTGAAGTCCTCATTAGTGAGGCCGGGATC CAAAGCGGAACCATGACTTTCTTCCCAAGCTCCCCACTCGATACCTTGGCTCTTACCGGAGAGGAT CTCAGAGATGGGCCTAGCGTTAGTGTTAAGGCTTGGGGCCTTCAGTGCCGCAGGAATCGAACAACA GCCAGTAAGAGGTTCCGCGCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC ATGTCTTACTTGAAAATTTCCGCTTTCCTTTCAGTTTTGTCCGTCGCCTTGGCCGACCAACGAATC TCTGTCACCGTTGTTGGTGATGGTATCAACACAGGACTGAGATCCGGAGGGTCGCATTTTGAGGCT GGACCAAATGCTGCTGGTACCCCCTTAGATTTGATCCTGTATGAGCCGTCTGGTTTCTTGGTAGAT GCAGCAGATGCTTCCAAGTACGTTGGTTGGGATGTTGCAGCTGGCACTTCTTTGACTTCTTTGCCA CCAGACCAAGGAGGCAAAGATTGGGGAATCGTTGCCGGTAACCTCAGATTCAACGTTGGAGGTACT ACATTCTATGCTTGTGAGACTAGGACCGGTGTTTGGGAAGTAAAGAGTTACGAAGCTAGTGGATGC AACGCTGTGGTGCTTTCCGTAGCTAGTCACCCAGTTCCTTCTTCCAGTTCTTCCAGTTCTTCCCAT GCCCCAACTTCCTCTGTTCCATCTACTTCGTCTCATGTGAGCCCAACTACCACTCAACCTCCTCAC ACAACCAGTTCTCACACCATCCACACATCAACTACCTTGACGACATCAGGTAGGAATGACTCGAAC CACTCCAACCATACCATCCCACCAGTTCCAACCGGTGCCGCTATGGGAGTCTCTAGCAACTATGGT TTGTTGGTTGCAGCTGGAATTGCCGCCGCTGCTTTGTTATAAGCGGCCGCGAATTAATTCGCCTTA GACA
SEQUENCE LISTING
<110> Shanghai Dragon Biotechnology Co., ltd
<120> a method for preparing pichia pastoris displaying fructosyl transferase on surface
<130> 2021
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 1575
<212> DNA
<213> Artificial sequence
<400> 1
atgtatatca aaccattcat cctacctgcc cttgcggcgg ttgcgcaagc tgccagctat 60
tcgggagacc ttcggcctca aactcactcc tctccacctt ccaatttcac gaacgatcca 120
agcggtctct tctatgatag caagaggggc gtgtatcact tatactatca gtataagcct 180
acagcgacag tctctgggaa tcagcactgg ggtcatgcca ccagccctga tctatcgcac 240
tggacgaatc aacgtatcgc cctcgctggg gataagccag aggagtatat cttctcaggc 300
tctgctgtcg tggacagcaa caacagcaca gcaatatttc cggaacagga cgatggcgtc 360
atagccatct acacagtaaa taccccgaca ctggaaacac atctcattcc ttcttctagc 420
aatggtggat acactttcac caagtatgag aacaacgagg tcatcgacat tggctcaaag 480
acgttccgtg acccggaggt ggtgcggcat cctgaaacta cgcaatgggt gatgacactt 540
gcctatgcgg agaacttggt catccgattt tatacctcgc ccaatctcaa agactggtcg 600
actcggacgt atatcacgct ggaacggcag acgggcgacc aattcgaatg ccccaacttg 660
gtcaagtttt cggtgactca cgctgagtct gaggagacgt gcatgtacgt gcttttcctt 720
tccgtgtcca ctggcgctcc tcttggaggt tcgcgaacac tttatgtgga cggcgatttc 780
tacggcagcc attacacctc tgaagttgct cagaagatac tatttgaatt ctccaagtac 840
aactatccgg ctcagtgcta ttctggtatt cctgagaacg agcctcctgt ctccatcggg 900
tggcctagta cgtgccatta cacggaagaa gtcccaactg gcccgcttga aggctggagg 960
agcgccatga ctttacctag aacgcagacc ataagcaacg tcaacggagt ctggactgtg 1020
actcacagcc cgtttgaaag cccatcggat cctaagggca gacagcttgt gagcaagagt 1080
gtccactctg gggatgtgaa gacgaagtta tccggggtcc catcgaatgc gatatatttc 1140
aatgtgacac tgaagggcat cgatgttacc aggtctaccg gacgcctaaa cttcagcttt 1200
tcctcctctg tatcgggtga attcctcgac ggaggggtgt ctctggatga tagttctctc 1260
tggatgacca gagctggatc ccatctattc acgtttgaag ctagtggaaa ttatacatca 1320
tcttctacaa catctatcac cccctttggc aacagcatat tcaccttctc gggcgttatt 1380
gaccgctctg tgtttgaagt cctcattagt gaggccggga tccaaagcgg aaccatgact 1440
ttcttcccaa gctccccact cgataccttg gctcttaccg gagaggatct cagagatggg 1500
cctagcgtta gtgttaaggc ttggggcctt cagtgccgca ggaatcgaac aacagccagt 1560
aagaggttcc gcgcc 1575
<210> 2
<211> 702
<212> DNA
<213> Artificial sequence
<400> 2
atgtcttact tgaaaatttc cgctttcctt tcagttttgt ccgtcgcctt ggccgaccaa 60
cgaatctctg tcaccgttgt tggtgatggt atcaacacag gactgagatc cggagggtcg 120
cattttgagg ctggaccaaa tgctgctggt acccccttag atttgatcct gtatgagccg 180
tctggtttct tggtagatgc agcagatgct tccaagtacg ttggttggga tgttgcagct 240
ggcacttctt tgacttcttt gccaccagac caaggaggca aagattgggg aatcgttgcc 300
ggtaacctca gattcaacgt tggaggtact acattctatg cttgtgagac taggaccggt 360
gtttgggaag taaagagtta cgaagctagt ggatgcaacg ctgtggtgct ttccgtagct 420
agtcacccag ttccttcttc cagttcttcc agttcttccc atgccccaac ttcctctgtt 480
ccatctactt cgtctcatgt gagcccaact accactcaac ctcctcacac aaccagttct 540
cacaccatcc acacatcaac taccttgacg acatcaggta ggaatgactc gaaccactcc 600
aaccatacca tcccaccagt tccaaccggt gccgctatgg gagtctctag caactatggt 660
ttgttggttg cagctggaat tgccgccgct gctttgttat aa 702
<210> 3
<211> 51
<212> DNA
<213> Artificial sequence
<400> 3
ggcggcggcg gcagcggcgg cggcggcagc agcagcggcg gcggcggcag c 51
<210> 4
<211> 2368
<212> DNA
<213> Artificial sequence
<400> 4
gagaggctga agcttacgta gaattcatgt atatcaaacc attcatccta cctgcccttg 60
cggcggttgc gcaagctgcc agctattcgg gagaccttcg gcctcaaact cactcctctc 120
caccttccaa tttcacgaac gatccaagcg gtctcttcta tgatagcaag aggggcgtgt 180
atcacttata ctatcagtat aagcctacag cgacagtctc tgggaatcag cactggggtc 240
atgccaccag ccctgatcta tcgcactgga cgaatcaacg tatcgccctc gctggggata 300
agccagagga gtatatcttc tcaggctctg ctgtcgtgga cagcaacaac agcacagcaa 360
tatttccgga acaggacgat ggcgtcatag ccatctacac agtaaatacc ccgacactgg 420
aaacacatct cattccttct tctagcaatg gtggatacac tttcaccaag tatgagaaca 480
acgaggtcat cgacattggc tcaaagacgt tccgtgaccc ggaggtggtg cggcatcctg 540
aaactacgca atgggtgatg acacttgcct atgcggagaa cttggtcatc cgattttata 600
cctcgcccaa tctcaaagac tggtcgactc ggacgtatat cacgctggaa cggcagacgg 660
gcgaccaatt cgaatgcccc aacttggtca agttttcggt gactcacgct gagtctgagg 720
agacgtgcat gtacgtgctt ttcctttccg tgtccactgg cgctcctctt ggaggttcgc 780
gaacacttta tgtggacggc gatttctacg gcagccatta cacctctgaa gttgctcaga 840
agatactatt tgaattctcc aagtacaact atccggctca gtgctattct ggtattcctg 900
agaacgagcc tcctgtctcc atcgggtggc ctagtacgtg ccattacacg gaagaagtcc 960
caactggccc gcttgaaggc tggaggagcg ccatgacttt acctagaacg cagaccataa 1020
gcaacgtcaa cggagtctgg actgtgactc acagcccgtt tgaaagccca tcggatccta 1080
agggcagaca gcttgtgagc aagagtgtcc actctgggga tgtgaagacg aagttatccg 1140
gggtcccatc gaatgcgata tatttcaatg tgacactgaa gggcatcgat gttaccaggt 1200
ctaccggacg cctaaacttc agcttttcct cctctgtatc gggtgaattc ctcgacggag 1260
gggtgtctct ggatgatagt tctctctgga tgaccagagc tggatcccat ctattcacgt 1320
ttgaagctag tggaaattat acatcatctt ctacaacatc tatcaccccc tttggcaaca 1380
gcatattcac cttctcgggc gttattgacc gctctgtgtt tgaagtcctc attagtgagg 1440
ccgggatcca aagcggaacc atgactttct tcccaagctc cccactcgat accttggctc 1500
ttaccggaga ggatctcaga gatgggccta gcgttagtgt taaggcttgg ggccttcagt 1560
gccgcaggaa tcgaacaaca gccagtaaga ggttccgcgc cggcggcggc ggcagcggcg 1620
gcggcggcag cggcggcggc ggcagcatgt cttacttgaa aatttccgct ttcctttcag 1680
ttttgtccgt cgccttggcc gaccaacgaa tctctgtcac cgttgttggt gatggtatca 1740
acacaggact gagatccgga gggtcgcatt ttgaggctgg accaaatgct gctggtaccc 1800
ccttagattt gatcctgtat gagccgtctg gtttcttggt agatgcagca gatgcttcca 1860
agtacgttgg ttgggatgtt gcagctggca cttctttgac ttctttgcca ccagaccaag 1920
gaggcaaaga ttggggaatc gttgccggta acctcagatt caacgttgga ggtactacat 1980
tctatgcttg tgagactagg accggtgttt gggaagtaaa gagttacgaa gctagtggat 2040
gcaacgctgt ggtgctttcc gtagctagtc acccagttcc ttcttccagt tcttccagtt 2100
cttcccatgc cccaacttcc tctgttccat ctacttcgtc tcatgtgagc ccaactacca 2160
ctcaacctcc tcacacaacc agttctcaca ccatccacac atcaactacc ttgacgacat 2220
caggtaggaa tgactcgaac cactccaacc ataccatccc accagttcca accggtgccg 2280
ctatgggagt ctctagcaac tatggtttgt tggttgcagc tggaattgcc gccgctgctt 2340
tgttataacc tagggcggcc gcgaatta 2368
<210> 5
<211> 1634
<212> DNA
<213> Artificial sequence
<400> 5
gagaggctga agcttacgta gaattcatgt atatcaaacc attcatccta cctgcccttg 60
cggcggttgc gcaagctgcc agctattcgg gagaccttcg gcctcaaact cactcctctc 120
caccttccaa tttcacgaac gatccaagcg gtctcttcta tgatagcaag aggggcgtgt 180
atcacttata ctatcagtat aagcctacag cgacagtctc tgggaatcag cactggggtc 240
atgccaccag ccctgatcta tcgcactgga cgaatcaacg tatcgccctc gctggggata 300
agccagagga gtatatcttc tcaggctctg ctgtcgtgga cagcaacaac agcacagcaa 360
tatttccgga acaggacgat ggcgtcatag ccatctacac agtaaatacc ccgacactgg 420
aaacacatct cattccttct tctagcaatg gtggatacac tttcaccaag tatgagaaca 480
acgaggtcat cgacattggc tcaaagacgt tccgtgaccc ggaggtggtg cggcatcctg 540
aaactacgca atgggtgatg acacttgcct atgcggagaa cttggtcatc cgattttata 600
cctcgcccaa tctcaaagac tggtcgactc ggacgtatat cacgctggaa cggcagacgg 660
gcgaccaatt cgaatgcccc aacttggtca agttttcggt gactcacgct gagtctgagg 720
agacgtgcat gtacgtgctt ttcctttccg tgtccactgg cgctcctctt ggaggttcgc 780
gaacacttta tgtggacggc gatttctacg gcagccatta cacctctgaa gttgctcaga 840
agatactatt tgaattctcc aagtacaact atccggctca gtgctattct ggtattcctg 900
agaacgagcc tcctgtctcc atcgggtggc ctagtacgtg ccattacacg gaagaagtcc 960
caactggccc gcttgaaggc tggaggagcg ccatgacttt acctagaacg cagaccataa 1020
gcaacgtcaa cggagtctgg actgtgactc acagcccgtt tgaaagccca tcggatccta 1080
agggcagaca gcttgtgagc aagagtgtcc actctgggga tgtgaagacg aagttatccg 1140
gggtcccatc gaatgcgata tatttcaatg tgacactgaa gggcatcgat gttaccaggt 1200
ctaccggacg cctaaacttc agcttttcct cctctgtatc gggtgaattc ctcgacggag 1260
gggtgtctct ggatgatagt tctctctgga tgaccagagc tggatcccat ctattcacgt 1320
ttgaagctag tggaaattat acatcatctt ctacaacatc tatcaccccc tttggcaaca 1380
gcatattcac cttctcgggc gttattgacc gctctgtgtt tgaagtcctc attagtgagg 1440
ccgggatcca aagcggaacc atgactttct tcccaagctc cccactcgat accttggctc 1500
ttaccggaga ggatctcaga gatgggccta gcgttagtgt taaggcttgg ggccttcagt 1560
gccgcaggaa tcgaacaaca gccagtaaga ggttccgcgc cggcggcggc ggcagcggcg 1620
gcggcggcag cagc 1634
<210> 6
<211> 768
<212> DNA
<213> Artificial sequence
<400> 6
gcggcggcgg cggcagcagc agcggcggcg gcggcagcat gtcttacttg aaaatttccg 60
ctttcctttc agttttgtcc gtcgccttgg ccgaccaacg aatctctgtc accgttgttg 120
gtgatggtat caacacagga ctgagatccg gagggtcgca ttttgaggct ggaccaaatg 180
ctgctggtac ccccttagat ttgatcctgt atgagccgtc tggtttcttg gtagatgcag 240
cagatgcttc caagtacgtt ggttgggatg ttgcagctgg cacttctttg acttctttgc 300
caccagacca aggaggcaaa gattggggaa tcgttgccgg taacctcaga ttcaacgttg 360
gaggtactac attctatgct tgtgagacta ggaccggtgt ttgggaagta aagagttacg 420
aagctagtgg atgcaacgct gtggtgcttt ccgtagctag tcacccagtt ccttcttcca 480
gttcttccag ttcttcccat gccccaactt cctctgttcc atctacttcg tctcatgtga 540
gcccaactac cactcaacct cctcacacaa ccagttctca caccatccac acatcaacta 600
ccttgacgac atcaggtagg aatgactcga accactccaa ccataccatc ccaccagttc 660
caaccggtgc cgctatggga gtctctagca actatggttt gttggttgca gctggaattg 720
ccgccgctgc tttgttataa gcggccgcga attaattcgc cttagaca 768
<210> 7
<211> 2376
<212> DNA
<213> Artificial sequence
<400> 7
gagaggctga agcttacgta gaattcatgt atatcaaacc attcatccta cctgcccttg 60
cggcggttgc gcaagctgcc agctattcgg gagaccttcg gcctcaaact cactcctctc 120
caccttccaa tttcacgaac gatccaagcg gtctcttcta tgatagcaag aggggcgtgt 180
atcacttata ctatcagtat aagcctacag cgacagtctc tgggaatcag cactggggtc 240
atgccaccag ccctgatcta tcgcactgga cgaatcaacg tatcgccctc gctggggata 300
agccagagga gtatatcttc tcaggctctg ctgtcgtgga cagcaacaac agcacagcaa 360
tatttccgga acaggacgat ggcgtcatag ccatctacac agtaaatacc ccgacactgg 420
aaacacatct cattccttct tctagcaatg gtggatacac tttcaccaag tatgagaaca 480
acgaggtcat cgacattggc tcaaagacgt tccgtgaccc ggaggtggtg cggcatcctg 540
aaactacgca atgggtgatg acacttgcct atgcggagaa cttggtcatc cgattttata 600
cctcgcccaa tctcaaagac tggtcgactc ggacgtatat cacgctggaa cggcagacgg 660
gcgaccaatt cgaatgcccc aacttggtca agttttcggt gactcacgct gagtctgagg 720
agacgtgcat gtacgtgctt ttcctttccg tgtccactgg cgctcctctt ggaggttcgc 780
gaacacttta tgtggacggc gatttctacg gcagccatta cacctctgaa gttgctcaga 840
agatactatt tgaattctcc aagtacaact atccggctca gtgctattct ggtattcctg 900
agaacgagcc tcctgtctcc atcgggtggc ctagtacgtg ccattacacg gaagaagtcc 960
caactggccc gcttgaaggc tggaggagcg ccatgacttt acctagaacg cagaccataa 1020
gcaacgtcaa cggagtctgg actgtgactc acagcccgtt tgaaagccca tcggatccta 1080
agggcagaca gcttgtgagc aagagtgtcc actctgggga tgtgaagacg aagttatccg 1140
gggtcccatc gaatgcgata tatttcaatg tgacactgaa gggcatcgat gttaccaggt 1200
ctaccggacg cctaaacttc agcttttcct cctctgtatc gggtgaattc ctcgacggag 1260
gggtgtctct ggatgatagt tctctctgga tgaccagagc tggatcccat ctattcacgt 1320
ttgaagctag tggaaattat acatcatctt ctacaacatc tatcaccccc tttggcaaca 1380
gcatattcac cttctcgggc gttattgacc gctctgtgtt tgaagtcctc attagtgagg 1440
ccgggatcca aagcggaacc atgactttct tcccaagctc cccactcgat accttggctc 1500
ttaccggaga ggatctcaga gatgggccta gcgttagtgt taaggcttgg ggccttcagt 1560
gccgcaggaa tcgaacaaca gccagtaaga ggttccgcgc cggcggcggc ggcagcggcg 1620
gcggcggcag cggcggcggc ggcagcatgt cttacttgaa aatttccgct ttcctttcag 1680
ttttgtccgt cgccttggcc gaccaacgaa tctctgtcac cgttgttggt gatggtatca 1740
acacaggact gagatccgga gggtcgcatt ttgaggctgg accaaatgct gctggtaccc 1800
ccttagattt gatcctgtat gagccgtctg gtttcttggt agatgcagca gatgcttcca 1860
agtacgttgg ttgggatgtt gcagctggca cttctttgac ttctttgcca ccagaccaag 1920
gaggcaaaga ttggggaatc gttgccggta acctcagatt caacgttgga ggtactacat 1980
tctatgcttg tgagactagg accggtgttt gggaagtaaa gagttacgaa gctagtggat 2040
gcaacgctgt ggtgctttcc gtagctagtc acccagttcc ttcttccagt tcttccagtt 2100
cttcccatgc cccaacttcc tctgttccat ctacttcgtc tcatgtgagc ccaactacca 2160
ctcaacctcc tcacacaacc agttctcaca ccatccacac atcaactacc ttgacgacat 2220
caggtaggaa tgactcgaac cactccaacc ataccatccc accagttcca accggtgccg 2280
ctatgggagt ctctagcaac tatggtttgt tggttgcagc tggaattgcc gccgctgctt 2340
tgttataagc ggccgcgaat taattcgcct tagaca 2376
Claims (1)
1. A method for preparing Pichia pastoris with surface display of fructosyl transferase, comprising the steps of:
step 1: synthesis of fusion sequences
Wherein, the fructosyl transferase gene sequence shown in SEQ ID No.1 and the pichia pastoris anchored protein gene sequence shown in SEQ ID No.2 are fused through a connecting peptide sequence shown in SEQ ID No.3 to obtain an improved fusion sequence of the fructosyl transferase gene and the pichia pastoris anchored protein gene, and the fused gene plus carrier homologous arm sequence is shown in SEQ ID No.4 or SEQ ID No. 7;
step 2: construction of Pichia expression vector
Connecting or recombining the fusion sequence with a enzyme digestion linearized pichia pastoris expression vector to obtain a recombinant expression vector;
and step 3: transformation of Pichia pastoris
Transforming the constructed recombinant expression vector into a pichia pastoris competent cell;
and 4, step 4: screening for multicopy positive clones
Screening out positive pichia pastoris containing multi-copy fusion genes through high resistance;
and 5: obtaining pichia pastoris displaying fructosyl transferase on surface
And (3) obtaining the pichia pastoris with the fructosyl transferase displayed on the surface after methanol induction expression.
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