Pichia pastoris indirect surface display system based on TRAP protein scaffold and construction method and application thereof

Abstract

The invention discloses a Pichia pastoris indirect surface display system based on a TRAP protein scaffold, which is characterized in that Sed1p protein from saccharomyces cerevisiae is used as anchoring protein, and tetrapeptide repeat affinity protein TRAP is fixed on the anchoring protein on the surface of a Pichia pastoris cell to obtain the Pichia pastoris indirect surface display system based on the TRAP protein scaffold. The target protein with the carboxyl end fused with the peptide MEEVV or the peptide MRRVW is indirectly displayed on the surface of the pichia pastoris cell through specific binding with the tetrapeptide repeat affinity protein TRAP. The xylanase and the beta-xylosidase are indirectly displayed on the surface of the pichia pastoris cell by utilizing the surface display system, so that the pichia pastoris surface display system has the catalytic capabilities of the xylanase and the beta-xylosidase, can perform a synergistic effect to degrade the zelkova xylan, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a Pichia pastoris indirect surface display system based on a TRAP protein scaffold, its construction method, and its application. Background Technology

[0002] In the field of biocatalysis, yeast surface display technology, as an emerging bioengineering technique, anchors target proteins or heteropeptides to the cell surface through genetic engineering. Compared to intracellular expression systems, surface display technology has become a research hotspot due to its advantages such as simplified preparation process, improved stability, enhanced catalytic efficiency, and reusability and regeneration capabilities in repeated batch reactions.

[0003] Based on different surface display methods, yeast can be divided into direct surface display and indirect surface display. Direct surface display refers to the direct fusion of the target protein with the cell wall anchoring sequence. After expression, the fused protein is directly localized to the cell surface through covalent or non-covalent interactions. Indirect surface display, on the other hand, is mediated by scaffold proteins or other mediator molecules. The target protein first binds to the mediator, and then the mediator interacts with the cell surface anchoring elements to achieve localization. However, direct cell surface display cannot effectively utilize cell surface space, and the accessibility of substrates or intermediates is affected when multiple enzymes collaborate. Protein scaffold-mediated display systems can compensate for the shortcomings of direct display systems, shortening substrate transfer distances and enhancing the proximity effect between enzymes. Currently, protein scaffold-based display systems have applications in medicine, fuel, and agriculture. For example, Fan et al. designed a dual-microscaffold protein system to secrete and express cellulase derived from Clostridium mesophilum, enabling synergistic production of bioethanol through cellulose hydrolysis and yeast fermentation.

[0004] In existing indirect surface display systems, the ligand peptide sequences that specifically bind to mediator molecules such as scaffold proteins are relatively long, such as Cohesin (approximately 150 amino acids) in the fibrosome scaffold system and SpyTag protein (13 amino acids) in the SpyTag / SpyCatcher system. Long ligand peptides can easily lead to the inactivation of display enzymes.

[0005] Therefore, it is particularly important to construct a new, efficient, and stable indirect surface display system. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a Pichia pastoris indirect surface display system based on TRAP protein scaffold, which addresses the shortcomings of the prior art.

[0007] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned Pichia pastoris indirect surface display system based on TRAP protein scaffold.

[0008] Another technical problem to be solved by the present invention is to provide a recombinant Pichia pastoris that displays xylanase and / or β-xylosidase on the surface through the aforementioned TRAP protein scaffold-based Pichia pastoris indirect surface display system.

[0009] The final technical problem to be solved by this invention is to provide the application of recombinant Pichia pastoris with the above-mentioned surface display of xylanase and / or β-xylosidase.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] A Pichia pastoris indirect surface display system based on a TRAP protein scaffold, wherein the Pichia pastoris indirect surface display system uses Sed1p protein derived from Saccharomyces cerevisiae as an anchor protein, and fixes the tetrapeptide repeat affinity protein TRAP on the anchor protein on the surface of Pichia pastoris cells, and the target protein is indirectly displayed on the surface of Pichia pastoris cells by specifically binding to the tetrapeptide repeat affinity protein TRAP.

[0012] The amino acid sequence of the Sed1p protein is shown in SEQ ID NO:1; the amino acid sequence of the tetratricopeptide repeat affinity protein TRAP is shown in SEQ ID NO:2. The tetratricopeptide repeat affinity protein TRAP is a combination of three tetratricopeptide repeat affinity proteins, TRAP1, TRAP3, and TRAP2, and the nucleotide sequences of TRAP1, TRAP3, and TRAP2 are shown in SEQ ID NO:7 to 9, respectively.

[0013] Among them, the Pichia pastoris is Komagataella phaffii PSCS00 is a strain based on Pichia pastoris GS115. Using a CRISPR-Cas9 gene editing plasmid, genes in the Pichia pastoris GS115 genome were knocked out. ku70 (Genes involved in DNA double-strand break repair) and mph1 (An ATP-dependent DNA helicase gene), and in the Pichia pastoris GS115 genome PNSI-1 The site was inserted with a gene rad52 Expression cassette P of (homologous recombination-related genes) GAP - rad52 -T AOXThe recombinant Pichia pastoris obtained was constructed using a method described in the literature "Zhou C, Zhu Y, Ren P, et al. Construction of an efficient enzyme-cell@material biocatalyst through the biofilm immobilization of..." Komagataella phaffii The paper is published in Bioresource Technology 428 (2025) 132460” for continuous biocatalysis.

[0014] The target protein has a 5-amino acid peptide MEEVV (M1) or MRRVW (M3) fused to its carboxyl terminus; the amino acid sequences of the peptides MEEVV and MRRVW are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0015] Preferably, the nucleotide sequences of the genes encoding the peptides MEEVV and MRRVW are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0016] Preferably, the target protein is a green fluorescent protein GFP, a red fluorescent protein mRudy2, a xylanase, or a β-xylosidase, in which a peptide MEEVV (M1) or MRRVW (M3) of 5 amino acids is fused to the carboxyl terminus.

[0017] Preferably, the target protein is a green fluorescent protein GFP (GFP-M1) with M1 fused to its carboxyl terminus, a red fluorescent protein mRudy2 (mRudy2-M3) with M3 fused to its carboxyl terminus, a xylanase (xynANP-M1 or xynANP-M3) with M1 or M3 fused to its carboxyl terminus, or a β-xylosidase (Hixyl43A-M1 or Hixyl43A-M3) with M1 or M3 fused to its carboxyl terminus.

[0018] Preferably, the nucleotide sequences of the encoding genes of the target proteins GFP-M1, mRudy2-M3, xynANP-M1, xynANP-M3, Hixyl43A-M1 and Hixyl43A-M3 are shown in SEQ ID NO:11 ~ 16, respectively.

[0019] The above-mentioned method for constructing the Pichia pastoris indirect surface display system based on the TRAP protein scaffold includes the following steps:

[0020] Step 1: Fuse the gene encoding the tetrapeptide repeat affinity protein TRAP with the gene encoding the Sed1p protein to obtain a fusion gene. Trap-sed1 The fusion gene Trap-sed1 The Pichia pastoris genome was inserted to obtain recombinant Pichia pastoris PSCS14;

[0021] Step 2: Insert the gene encoding the target protein into the genome of the recombinant Pichia pastoris PSCS14 obtained in Step 1 to obtain the recombinant Pichia pastoris XMHM, which can indirectly display the target protein, also known as the Pichia pastoris indirect surface display system.

[0022] Preferably, the method for constructing the Pichia pastoris indirect surface display system based on the TRAP protein scaffold includes the following steps:

[0023] Step 1: Fuse the gene encoding the tetrapeptide repeat affinity protein TRAP with the gene encoding the Sed1p protein to obtain a fusion gene. Trap-sed1 Construct the fusion gene Trap-sed1 The expression cassette contains the fusion gene. Trap- sed1 The expression cassette was inserted into the genome of the Pichia pastoris using CRISPR / Cas9 gene editing technology to obtain recombinant Pichia pastoris PSCS14;

[0024] Step 2: Construct an expression cassette for the gene encoding the target protein, and insert it into the genome of the recombinant Pichia pastoris PSCS14 obtained in Step 1 using CRISPR / Cas9 gene editing technology to obtain a recombinant Pichia pastoris that can indirectly display the target protein, i.e., the Pichia pastoris indirect surface display system.

[0025] In step 1, the nucleotide sequence of the gene encoding the tetrapeptide repeat affinity protein TRAP is shown in SEQ ID NO:5; the nucleotide sequence of the gene encoding the Sed1p protein is shown in SEQ ID NO:6.

[0026] Preferably, in step 1, the fusion gene Trap-sed1 The nucleotide sequence is shown in SEQ ID NO:10.

[0027] Preferably, the fusion gene Trap-sed1 The promoters in both the expression cassette and the expression cassette of the gene encoding the target protein are GAP promoters (P...). GAP ).

[0028] A recombinant Pichia pastoris, wherein the recombinant Pichia pastoris displays target proteins xylanase and / or β-xylosidase on its surface through the aforementioned TRAP protein scaffold-based Pichia pastoris indirect surface display system.

[0029] The xylanase has the peptide MEEVV or MRRVW fused to its carboxyl terminus; the β-xylosidase has the peptide MEEVV or MRRVW fused to its carboxyl terminus.

[0030] In the described Pichia pastoris indirect surface display system based on the TRAP protein scaffold, TRAP1 and TRAP3 in the TRAP protein scaffold can bind with high affinity to the 5-amino acid ligand peptides MEEVV (M1) and MRRVW (M3), respectively. Based on this principle, on the Pichia pastoris genetically engineered strain that overexpresses the TRAP protein scaffold, xylanase with the carboxyl terminus fused with the ligand peptides M1 or M3 and / or β-xylosidase with the carboxyl terminus fused with the ligand peptides M1 or M3 are further overexpressed, thereby indirectly displaying xylanase and / or β-xylosidase on the surface of Pichia pastoris cells.

[0031] The present invention also provides the application of the above-mentioned recombinant Pichia pastoris with surface-displaying xylanase and / or β-xylosidase in the degradation of xylan.

[0032] During the fermentation culture of the above-mentioned recombinant Pichia pastoris, the TRAP protein scaffold on the surface of the recombinant Pichia pastoris cells undergoes high-affinity specific dual-enzyme self-assembly with the ligand peptides M1 and / or M3 in the target proteins (xylanase and / or β-xylosidase). After the fermentation culture is completed, the fermentation broth is centrifuged, and the Pichia pastoris cells displaying the enzymes on the surface are collected for catalytic degradation of xylan.

[0033] Preferably, the fermentation culture is carried out in a free fermentation culture at a temperature of 28-30℃ and a rotation speed of 220-250 rpm for 90-150 h; when carrying out the catalytic degradation of xylan, the working temperature is 45-65℃.

[0034] Preferably, the xylan is beech xylan, corn cob xylan, wheat xylan, beet xylan, and xylan contained in sugarcane bagasse and filter paper pulp.

[0035] Beneficial effects:

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The Pichia pastoris indirect surface display system based on the TRAP protein scaffold of the present invention involves fusing the TRAP protein scaffold with the anchoring protein Sed1p for extracellular secretion and immobilization on the surface of Pichia pastoris cells to form a surface protein scaffold. Then, utilizing the high affinity and specific binding of TRAP1 in the TRAP protein scaffold to the target protein ligand M1 or TRAP3 to the ligand M3, the target protein expressed and secreted by the Pichia pastoris cells self-assembles on the yeast surface, thereby achieving indirect surface display of the target protein. This invention utilizes this surface display system to indirectly display xylanase and β-xylosidase on the surface of Pichia pastoris cells, giving them both xylanase and β-xylosidase catalytic capabilities, enabling synergistic degradation of beech xylan, and showing promising application prospects.

[0038] (2) In the Pichia pastoris indirect surface display system based on the TRAP protein scaffold of the present invention, the ligand peptides M1 and M3 that bind specifically to the TRAP protein scaffold with high affinity contain only 5 amino acid sequences, which are smaller than the sequence lengths of Cohesin (about 150 amino acids) in the fibrosome scaffold system and SpyTag protein (13 amino acids) in the SpyTag / SpyCatcher system in the prior art. The smaller ligand peptide units are more likely to retain the catalytic activity of the display enzyme. Attached Figure Description

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0040] Figure 1 The image shows an agarose gel electrophoresis diagram of the colony PCR of the successfully constructed Pichia pastoris genetically engineered strain PSCS14 in Example 1; lanes 1-4 represent different positive transformants of the recombinant strain, lane 5 represents the starting strain PSCS00, and lane M represents the DNA marker of DL5000.

[0041] Figure 2 The images show the results of laser confocal microscopy and flow cytometry analysis of the starting strain PSCS00 and the protein scaffold recombinant engineered strain PSCS14 in Example 2; where Figure A is a laser confocal fluorescence microscope image and Figure B is a flow cytometry histogram.

[0042] Figure 3 The image shows an agarose gel electrophoresis diagram of colony PCR of Pichia pastoris genetically engineered strains PSCS25 and PSCS26, which were successfully constructed in Example 3. Lane 1 is a positive transformant of recombinant strain PSCS25, lane 2 is a positive transformant of recombinant strain PSCS26, lane 3 is recombinant strain PSCS14, and lane M is the DNA marker of DL10000.

[0043] Figure 4 Figure 1 shows the results of laser confocal microscopy and flow cytometry analysis of recombinant strains PSCS14, PSCS25, and PSCS26 in Example 3; where Figure A is a laser confocal fluorescence microscope image and Figure B is a flow cytometry histogram.

[0044] Figure 5 The image shows the results of xylanase and β-xylosidase activity detection on the cell surface and supernatant of genetically engineered strains PSCS14, XM3HM1 and XM1HM3 in Example 4.

[0045] Figure 6 The amount of xylose produced during the catalytic hydrolysis of xylan by strains XM1HM3 and XM3HM1 in Example 5 is the amount accumulated over time. Detailed Implementation

[0046] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0047] The plasmids, restriction enzymes, high-fidelity enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are all commercial products. Specific procedures were performed according to the kit instructions. Other experimental consumables, unless otherwise specified, can be obtained commercially. The preparation of competent cells, colony PCR, nucleic acid agarose gel electrophoresis, electroporation, and extraction and preservation of yeast genome were performed according to standard methods.

[0048] The following genome-free integration expression system is a CRISPR-Cas9 gene-editing plasmid, donated by Professor Yongjin Zhou of the Synthetic Microbiology Research Group at the Dalian Institute of Chemical Physics, Chinese Academy of Sciences. It is described in the paper "Peng C, Xingpeng D, Xiaoyan W, et al. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast". Pichia pastoris [J]. NucleicAcids Research, 2021(13):13.” Published in

[0049] The sequencing of the following plasmids and DNA products was completed by Anhui General Biotechnology Co., Ltd.

[0050] The following examples illustrate the determination of xylose concentration in the catalytic system: 0.2 mL of the catalytic reaction solution was boiled at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was diluted a certain factor and filtered through a 0.22 μm filter membrane. The xylose concentration in the catalytic system was then detected using HPLC. HPLC detection method: differential refractive index detector; chromatographic column: BioRad HPX-87H (9 μm, 300 mm × 7.8 mm); column temperature: 60℃; mobile phase: 5 mmol / L H2SO4 aqueous solution; flow rate: 0.5 mL / min; injection volume: 5 μL.

[0051] Example 1: Construction of Pichia pastoris recombinant genetically engineered strain based on TRAP scaffold

[0052] First, an expression cassette containing the TRAP scaffold protein encoding gene was constructed; then, using Pichia pastoris PSCS00 as the starting strain, a fusion gene was integrated into the PNSⅣ-16 site of the genome using CRISPR / Cas9 gene editing technology. Trap-sed1 The expression box, and the specific experimental steps are as follows:

[0053] (1) Amplification of gene fragments: using plasmid pPIC9K- synthesized by GenScript. Trap-sed1 Using TRAP-F / Sed1-R as a template, the fusion gene was obtained by PCR amplification. Trap-sed1 The fusion gene Trap-sed1 The encoded TRAP protein contains a short Flag peptide tag at its N-terminus, which can specifically bind to fluorescently labeled antibodies. This fusion gene Trap- sed1 The nucleotide sequence is shown in SEQ ID NO:10; using the genome of strain PSCS00 as a template, primer pairs T PGK1 -F / T PGK1 -R was used for PCR amplification to obtain the terminator fragment T. PGK1 Using plasmid pGAPαA as a template, primer pairs P GAP -F / P GAP -R was used for PCR amplification to obtain the promoter fragment pGAPα; using the genome of strain PSCS00 as a template, PCR amplification was performed using primer pairs PNSⅣ16-Up-F / PNSⅣ16-Up-R and PNSⅣ16-Down(PGK1)-F / PNSⅣ16-Down(PGK1)-R to obtain the upper and lower homologous arm fragments PNSⅣ16-Up and PNSⅣ16-Down of the PNSⅣ16 site.

[0054] (2) Constructing fusion genes Trap-sed1 Expression cassette: containing fusion gene fragments Trap-sed1 Termination sub-fragment TPGK1 Using the promoter fragment pGAPα as a template, primer pairs P GAP -F / T PGK1 -R is used for overlap PCR amplification to obtain expression cassette fragments. P GAP -Trap-sed1-T PGK1 The fragments were purified by gel recovery.

[0055] (3) Constructing Donor DNA fragments: using expression cassette fragments P GAP -Trap-sed1-T PGK1 Using the homologous arm fragments PNSⅣ16-Up and PNSⅣ16-Down as templates, overlap PCR amplification was performed using primer pairs PNSⅣ16-Up-F and PNSⅣ16-Down(PGK1)-R to obtain the Donor DNA fragment, which was then purified by gel extraction.

[0056] (4) Pichia pastoris electroporation transformation: Take out the prepared Pichia pastoris PSCS00 competent cells from -80℃, add the tool plasmid pPICZ-Cas9-PNSⅣ-16 and the Donor DNA fragment constructed in step (3) at a ratio of 1 μg: 5 μg, and transfer to an electroporation cuvette and stand on ice for 5 min; perform electroporation transformation at 2000 V and 5 ms, and recover in a 30℃ incubator for 3 h; after recovery, take 100 μL of bacterial solution and spread it on a YPDZ resistant plate (containing 100 μg / mL bleomycin), and incubate at 30℃ for 3 d. Perform colony PCR verification on the single colonies that grow on the plate (using the primer pair PNSⅣ16-QF / PNSⅣ16-QR), screen positive transformants and perform gene sequencing.

[0057] Colony PCR validation results are as follows Figure 1 As shown, the original colony PCR band of the original starting strain Pichia pastoris PSCS00 at the PNSⅣ-16 site was approximately 1600 bp, while the colony PCR bands of the recombinant genetically engineered strains 1-4 transformants were approximately 4500 bp. Therefore, Example 1 successfully inserted a protein scaffold expression cassette at the PNSⅣ-16 site in the genome of strain PSCS00. P GAP -Trap-sed1-T PGK1 A recombinant Pichia pastoris strain, PSCS14, was obtained.

[0058] The primer sequences involved in the above experimental process are shown in Table 1.

[0059] Table 1 Primer Sequences

[0060]

[0061] Example 2: Laser confocal microscopy and flow cytometry verification of successful display of TRAP protein scaffolds on cell surfaces

[0062] The fusion gene in Example 1 above Trap-sed1 The encoded TRAP protein contains a short Flag peptide tag at its N-terminus, which can specifically bind to the fluorescently labeled antibody THE™ DYKDDDDK Tag Antibody [iFluor 647] (purchased from Nanjing Genscript Biotech Co., Ltd., catalog number A01811), indirectly labeling the TRAP protein scaffold on the surface of Pichia pastoris cells. The fluorescence signal carried by the antibody was then detected using flow cytometry and laser confocal microscopy to verify the display of the TRAP scaffold on the surface of Pichia pastoris cells. The specific operation procedure is as follows:

[0063] (1) Take 1 mL of Pichia pastoris PSCS14 and PSCS00 cell slurry cultured for 48 h, centrifuge at 1000 rpm for 10 min, collect the cell pellet, and use it for subsequent Flag tag staining.

[0064] (2) Wash the cell pellet twice with 1×PBS at pH=7.4, centrifuge at 1000 rpm for 10 min, and then resuspend in 1 mL PBS;

[0065] (3) Take 50 μL of the bacterial culture obtained in step (2), add 1 mL of 3% (3 g / 100 mL) BSA aqueous solution and incubate at 37℃ for 30 min;

[0066] (4) After incubation, add 200 μL of THE™ DYKDDDDK Tag Antibody [iFluor 647] which has been diluted 100 times with water, and incubate at 37°C for 1 h;

[0067] (5) After incubation, centrifuge at 1000 rpm for 10 min, discard the supernatant, add 1 mL of washing buffer (PBS containing 0.5% Tween 20) to the bacterial cells, centrifuge at 1000 rpm for 10 min twice, and discard the supernatant;

[0068] (6) Resuspend the bacterial cells obtained in step (6) with 500 μL PBS to obtain bacterial solution. Take 100 μL of bacterial solution and filter it through a 0.22 μm filter membrane for flow cytometry analysis.

[0069] (7) The bacterial culture obtained in step (7) is dropped onto a slide and observed under a laser confocal fluorescence microscope.

[0070] Laser confocal fluorescence microscopy image of Pichia pastoris cells as shown below Figure 2 As shown in Figure A, the original starting strain PSCS00 did not exhibit a fluorescent signal, while the recombinant genetically engineered strain PSCS14 showed a fluorescent signal on its cell surface. Therefore, the TRAP protein scaffold was successfully displayed on the yeast cell surface; flow cytometry results further confirmed this. Figure 2 (B) In the APC detection channel, the recombinant genetically engineered strain PSCS14 cell population shifted significantly to the right, with a positive cell rate of 32.2%. This result further confirms that the TRAP scaffold has been successfully displayed on the surface of yeast cells.

[0071] Example 3: Validating the feasibility of indirect display of TRAP scaffold proteins using GFP and mRuby2 fluorescent proteins.

[0072] Using the recombinant genetically engineered strain PSCS14 constructed in Example 1 as the starting strain, fluorescent protein expression cassettes were inserted into its genome. P GAP -gfp-M1-T AOX and P GAP - mRuby2-M3-T AOX To obtain Pichia pastoris genetically engineered recombinant strains PSCS25 and PSCS26, laser confocal microscopy and flow cytometry were then used to verify the fluorescent proteins indirectly displayed on the yeast cell surface. The specific steps are as follows:

[0073] (1) Amplification of gene fragments: target gene fragments gfp-M1 and mRuby2-M3 All were synthesized by Nanjing Genscript Biotech Co., Ltd. after codon optimization, and the target gene fragments were obtained by amplification using primer pairs GFP-F / GFP-M1-R and mRuby2-F / mRuby2-M3-R respectively; promoter fragments P GAP and termination sub-fragments T AOX Both were performed using plasmid pGAPZαA as a template, via primer pair P GAP -F / P GAP -R and T AOX -F / T AOX -R amplification was obtained; then the target gene fragment was used. gfp-M1 promoter P GAP and Termination Sub T AOX Using P as a template, primer pairs were used. GAP -F / T AOX -R was used for overlap PCR amplification to obtain fluorescent protein expression cassettes. PGAP -gfp-M1-T AOX ; with target gene fragment mRuby2-M3 promoter P GAP and Termination Sub T AOX Using P as a template, primer pairs were used. GAP -F / T AOX -R was used for overlap PCR amplification to obtain fluorescent protein expression cassettes. P GAP - mRuby2-M3-T AOX .

[0074] (2) Amplification of the upper and lower homologous arm fragments of the integration site PNSⅣ-2: Using the Pichia pastoris PSCS00 genome as a template, the upper and lower homologous arm fragments of the PNSⅣ-2 site were amplified using primer pairs PNSⅣ-2-UF / PNSⅣ-2-UR and PNSⅣ-2-DF / PNSⅣ-2-DR, respectively. PNSⅣ-2-Up and PNSⅣ-2-Down .

[0075] (3) Donor DNA fragment: Using the expression cassette fragment in (1) and the homologous arm fragment in (2) as templates, Donor DNA was obtained by Overlap PCR amplification using primer pair PNSⅣ-2-UF / PNSⅣ-2-DR.

[0076] (4) Pichia pastoris electroporation transformation: Pichia pastoris PSCS14 competent cells were taken out from -80℃ and the tool plasmid pPICZ-Cas9-PNSⅣ-2 and the Donor DNA fragment constructed in step (3) were added at a ratio of 1 μg: 5 μg. The cells were then transferred to an electroporation cuvette and placed on ice for 5 min. Electroporation transformation was performed at 2000 V and 5 ms, and the cells were incubated at 30℃ for 3 h. After incubation, 100 μL of bacterial culture was plated on YPDZ resistant plates (containing 100 μg / mL bleomycin) and incubated at 30℃ for 3 d. Single colonies growing on the plates were verified by colony PCR. Positive transformants were screened and gene sequencing was performed. Pichia pastoris recombinant strain PSCS25 (with an expression cassette inserted into the genome) was successfully obtained. P GAP - gfp-M1-T AOX ) and PSCS26 (genome insertion expression cassette) P GAP - mRuby2-M3-T AOX ).

[0077] (5) The fluorescent protein displayed on the cell surface scaffold of the recombinant Pichia pastoris strain was analyzed according to the laser confocal and flow cytometry operation methods in Example 2.

[0078] Colony PCR validation results are as follows Figure 3 As shown, the original band of the starting strain PSCS14 at the PNSIV-2 site was approximately 2100 bp, while the colony PCR bands of the indirectly displaying fluorescent protein recombinant genetically engineered strains PSCS25 and PSCS26 at this site were approximately 4000 bp. Therefore, this embodiment successfully inserted a fluorescent protein expression cassette at the PNSIV-2 site in the genome of strain PSCS14. P GAP -gfp-M1-T AOX and P GAP -mRuby2-M3-T AOX Two recombinant Pichia pastoris strains, PSCS25 and PSCS26, were successfully obtained.

[0079] The results of laser confocal and flow cytometry analysis showed that ( Figure 4 Since strain PSCS14 did not integrate the fluorescent protein gene, it did not fluoresce under a fluorescence microscope; however, bright green and red fluorescent signals were detected on the cell surfaces of the indirectly fluorescent protein recombinant genetically engineered strains PSCS25 and PSCS26, respectively. Figure 4 (A) indicates that GFP-M1 and mRudy2-M3 fluorescent proteins were successfully expressed and secreted in Pichia pastoris and targeted to the cell wall. Furthermore, flow cytometry analysis showed that 41% of cells in the recombinant genetically engineered strain PSCS25 successfully anchored to the green fluorescent protein GFP-M1, and 25.7% of cells in the recombinant genetically engineered strain PSCS26 successfully anchored to the red fluorescent protein mRudy2-M3. Figure 4 (B) This further verified the successful expression of the fluorescent protein expression cassette, and the directional binding of the two fluorescent proteins to the TRAP scaffold on the cell surface. Therefore, it is feasible to use TRAP scaffold proteins for indirect surface display of yeast.

[0080] The nucleotide sequences of the primers involved in this embodiment are shown in Table 2.

[0081] Table 2. Nucleotide sequences of primers

[0082]

[0083] Example 4: Construction and validation of a recombinant Pichia pastoris strain that indirectly displays bihemicellulase on its surface.

[0084] Using the recombinant genetically engineered strain PSCS14 constructed in Example 1 as the chassis host, xylanase expression cassettes were inserted into the PNSⅣ-2 site of its genome. P GAP -xynANP-M1-T AOX and P GAP -xynANP-M3-T AOX This process yielded Pichia pastoris recombinant strains XM1 and XM3. Then, using the constructed Pichia pastoris recombinant strain XM1 as a substrate, a β-xylosidase expression cassette was inserted into the PNSⅢ-5 site of its genome. P GAP -hixyl43A-M3-T AOX This led to the acquisition of the Pichia pastoris recombinant strain XM1HM3. Furthermore, using the recombinant strain XM3 as a substrate, a β-xylosidase expression cassette was inserted into the PNSⅢ-5 site of its genome. P GAP -hixyl43A-M1-T AOX This led to the acquisition of the Pichia pastoris recombinant strain XM3HM1. Target gene fragment xynANP-M1 , xynANP-M3, hixyl43A-M1, hixyl43A-M3 All samples were synthesized by Nanjing GenScript Biotech Co., Ltd. after codon optimization, and the target gene fragments were obtained by amplification using primer pairs xynANP-F / xynANP-M1-R, xynANP-F / xynANP-M3-R, Hixyl43A-F / Hixyl43A-M1-R, and Hixyl43A-F / Hixyl43A-M3-R, respectively. The construction methods of the xylanase expression cassette and β-xylosidase expression cassette, the electroporation transformation method of Pichia pastoris strain, and the specific steps of CRISPR / Cas9 gene editing in this embodiment are as described in Examples 1 and 3.

[0085] The primer sequences involved in this embodiment are shown in Table 3.

[0086] Table 3. Nucleotide sequences of primers.

[0087]

[0088] Note: The primer pairs PNSⅢ-5-UF / PNSⅢ-5-UR and PNSⅢ-5-DF / PNSⅢ-5-DR in the table are used to amplify the upper and lower homologous arm fragments of the PNSⅢ-5 insertion site in the Pichia pastoris PSCS00 genome.

[0089] The above-mentioned Pichia pastoris genetically engineered strains PSCS14, XM3HM1 and XM1HM3 were inoculated into YPD medium for shake-flask fermentation. Free fermentation culture was carried out at 30℃ and 250 rpm for 48 h. After centrifugation at 2000 rpm for 5 min, the supernatant and bacterial cells were collected. The bacterial cells were resuspended in 500 μL PBS to obtain bacterial solution.

[0090] Determination of xylanase activity:

[0091] The 3,5-dinitrosalicylic acid (DNS) reagent method was used to determine the enzyme activity: xylose produced by xylanase hydrolysis of beech xylan reacted with DNS reagent to form a colorimetric reaction. The absorbance was detected at 540 nm using ultraviolet spectrophotometry. The enzyme activity was quantitatively calculated by measuring the reducing xylose produced by the enzymatic reaction.

[0092] Add 5 μL of supernatant free enzyme (the supernatant above) and cell surface enzyme (the bacterial culture above) to 20 μL of potassium phosphate buffer (pH=6), then add 50 μL of 0.5% beech xylan solution (0.5 g dissolved in 100 mL of potassium phosphate buffer at pH 6.0). React accurately at 50℃ for 15 min, then add 100 μL of 3,5-dinitrosalicylic acid (DNS) reagent, mix thoroughly, and boil for 5 min. Cool the reaction solution to room temperature, then add 175 μL of distilled water to obtain the test sample. Two blank control groups were set up, using supernatant and bacterial culture inactivated at high temperature instead of the corresponding supernatant and bacterial culture. Other operations were the same as the experimental group. Zero the instrument using the blank control group, and detect the absorbance of the test sample at 540 nm using ultraviolet spectrophotometry. Under these conditions, the amount of enzyme required to release 1 μmol of xylose per minute is defined as 1 enzyme activity unit (U).

[0093] Preparation of xylose standard curve: Take 0, 1, 2, 3, 4, and 5 μL of 10 mg / mL xylose aqueous solution into clean test tubes, respectively. Make up the volume to 75 μL with potassium phosphate buffer (pH 6.0), then add 100 μL of DNS reagent, mix thoroughly, and boil for 5 min. Cool the reaction solution to room temperature, then add 175 μL of distilled water, shake well, and measure the absorbance at 540 nm. Construct a standard curve (Y=aX+b) with absorbance as the ordinate (Y-axis) and xylose content (μmol) as the abscissa (X-axis).

[0094] Determination of β-xylosidase activity:

[0095] Take two 200 μL 5 mM solutions pNPX (p-nitrophenyl-β-D-xyloside p-pyranoside) solution was preheated in a 50°C water bath for 3 min. 50 μL of the supernatant containing the free enzyme (the above supernatant) and the cell surface enzyme (the above bacterial culture) were added separately. The reaction was carried out at 50°C for 10 min, and the reaction was immediately terminated by adding 750 μL of 2 M Na₂CO₃ aqueous solution to obtain the test sample. Two blank control groups were set up, replacing the corresponding supernatant and bacterial culture with heat-inactivated supernatant and bacterial culture, with other operations the same as above. The blank control group was used to zero the instrument, and the OD value of the test sample at 410 nm was measured using a spectrophotometer. β-xylosidase activity unit (U) is defined as the rate at which 5 mM of β-xyloside is degraded per minute under the detection conditions. p NPX produces 1 μmol p The amount of enzyme required for NP.

[0096] p Preparation of NP standard curve: Take 0, 20, 40, 80, 120 and 160 μL of 1 mM standard curve. p Pour NP solution into a clean test tube, and bring the volume to 250 μL with citrate-Na₂HPO₄ buffer (pH 6.0). Then, add 750 μL of 2 M Na₂CO₃ aqueous solution to each tube to terminate the reaction. Shake well and measure the OD value at 410 nm using a spectrophotometer. Plot the absorbance value on the ordinate (Y-axis). p A standard curve (Y=aX+b) is constructed with the NP content (μmol) as the x-axis.

[0097] Test results as follows Figure 5 As shown, xylanase Xyn and β-xylosidase Hixyl43A were detected in both the cell pellets and supernatants of recombinant strains XM3HM1 and XM1HM3. Specifically, the cell surface activities of Xyn and Hixyl43A in recombinant strain XM3HM1 were 0.43 U / mL and 2.05 U / mL, respectively, while the free Xyn and Hixyl43A activities in the supernatant were 100.35 U / mL and 32.45 U / mL, respectively. Similarly, the cell surface activities of Xyn and Hixyl43A in recombinant strain XM1HM3 were 0.39 U / mL and 1.94 U / mL, respectively, while the free Xyn and Hixyl43A activities in the supernatant were 98.35 U / mL and 27.41 U / mL, respectively. Although the surface enzyme activities were lower than the free enzyme activities in the supernatant, the presence of certain enzyme catalytic activity on the cell surface demonstrates the successful realization of the enzyme display system based on the TRAP scaffold protein.

[0098] Example 5: Indirect demonstration and validation of the scale-up system for xylan degradation by the strain

[0099] β-xylosidase is an important member of the xylan hydrolytic enzyme system and can work synergistically with xylanase to hydrolyze xylan.

[0100] To investigate the catalytic efficiency of the engineered Pichia pastoris protein scaffold display strains in a 10 mL scale-up system, this example used recombinant Pichia pastoris strains XM1HM3 and XM3HM1 as target strains. The recombinant strains XM1HM3 and XM3HM1 were inoculated into 5 mL of YPD liquid medium and cultured overnight for activation. Then, at a 1% v / v inoculation rate, they were transferred to 50 mL of YPD liquid medium and cultured for another 48 h at 30°C and 250 rpm. 10 mL of XM1HM3 and XM3HM1 bacterial suspensions (OD200) were then collected. 600 The cells were centrifuged at 2000 rpm for 5 min, and then collected and added to 10 mL of 5 g / L beech xylan solution (phosphate buffer) for single-batch catalysis at 50 °C for 3 h. The xylose content of the product in the catalytic solution was determined by HPLC.

[0101] The results are as follows Figure 6 As shown, in a 10 mL scale-up catalytic system, strains XM1HM3 and XM3HM1 exhibited consistent catalytic processes for the catalytic system containing 5 g / L beech xylan: xylose was rapidly generated within the first 2 hours, with XM3HM1 showing a slightly higher xylose accumulation rate than XM1HM3; both strains reached peak xylose production around 2 hours, and the product content remained stable within the following 1 hour. These results confirm that the two indirectly exhibiting engineered bacteria possess highly efficient xylan degradation activity and good catalytic stability in the scale-up system.

[0102] This invention provides a Pichia pastoris indirect surface display system based on a TRAP protein scaffold, along with its construction method and application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A Pichia pastoris indirect surface display system based on a TRAP protein scaffold, characterized in that, The indirect surface display system of Pichia pastoris takes the Sed1p protein derived from Saccharomyces cerevisiae as an anchor protein, and a tetranectin TRAP is fixed on the anchor protein on the surface of the Pichia pastoris cells, and a target protein is indirectly displayed on the surface of the Pichia pastoris cells through specific binding with the tetranectin TRAP.

2. The Pichia pastoris indirect surface display system based on a TRAP protein scaffold according to claim 1, characterized in that, The amino acid sequence of the Sed1p protein is shown as SEQ ID NO:1, and the amino acid sequence of the tetranectin TRAP is shown as SEQ ID NO:

2.

3. The Pichia pastoris indirect surface display system based on a TRAP protein scaffold according to claim 1, characterized in that, The Pichia pastoris is Komagataella phaffii PSCS00.

4. The Pichia pastoris indirect surface display system based on a TRAP protein scaffold according to claim 1, characterized in that, The carboxy terminus of the target protein is fused with a peptide MEEVV or a peptide MRRVW, and the amino acid sequences of the peptide MEEVV and the peptide MRRVW are shown as SEQ ID NO:3 and SEQ ID NO:4, respectively.

5. The method for constructing a Pichia pastoris indirect surface display system based on a TRAP protein scaffold according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step 1, a fusion gene is obtained by fusing a coding gene of the tetrapeptide repeat protein TRAP with a coding gene of the Sed1p protein Trap-sed1 the fusion gene Trap-sed1 is inserted into the genome of the Pichia pastoris to obtain a recombinant Pichia pastoris PSCS14; In step 2, the coding gene of the target protein is inserted into the genome of the recombinant Pichia pastoris PSCS14 obtained in step 1.

6. The construction method of claim 5, wherein, In step 1, the nucleotide sequence of the coding gene of the tetranectin TRAP is shown as SEQ ID NO:5, and the nucleotide sequence of the coding gene of the Sed1p protein is shown as SEQ ID NO:

6.

7. A recombinant Pichia pastoris, characterized in that, The recombinant Pichia pastoris displays target proteins xylanase and / or β-xylosidase on the surface through the indirect surface display system of Pichia pastoris based on the TRAP protein scaffold according to any one of claims 1-4.

8. The recombinant Pichia pastoris of claim 7 is used for catalyzing degradation of xylan.

9. Use according to claim 8, characterized in that, The recombinant Pichia pastoris is fermented, the fermentation broth is centrifuged, and the cells are collected for catalyzing degradation of xylan.

10. Use according to claim 8, characterized in that, The fermentation is carried out in free state under the conditions of a temperature of 28-30℃ and a rotation speed of 220-250 rpm for 90-150 h, and when the degradation of xylan is carried out, the working temperature is 45-65℃. The fermentation is carried out in free state under the conditions of a temperature of 28-30℃ and a rotation speed of 220-250 rpm for 90-150 h, and when the degradation of xylan is carried out, the working temperature is 45-65℃.

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