Pichia pastoris high-secretion strain screening system and use method thereof

Abstract

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biotechnology, and particularly relates to a Pichia pastoris high-secreting strain screening system and a use method thereof. Background Art

[0002] In the field of synthetic biology and industrial biomanufacturing, the efficiency bottleneck of the "design-build-test" (DBTL) cycle has become a core obstacle to the industrialization process. Traditional screening technologies (such as shake flask culture combined with HPLC detection) are limited by the single processing volume (<10³ clones / time) and high costs (>50 US dollars / clone), resulting in the optimization cycle of industrial enzymes, biofuels or drug proteins as long as 6-12 months, which seriously delays product development. 6 -10 8 The optimization of complex metabolic pathways in large-scale mutant libraries. The existing screening methods have insufficient success rates, resulting in more than 90% of effective mutants being missed. Therefore, the development of ultra-high throughput (>10 6 The novel screening technology with high precision (R²≥0.90) and low cost (<0.1 USD / clone) has become a key breakthrough to break through the cycle rate limitation of DBTL and accelerate biomanufacturing from laboratory to 10,000-ton industrialization.

[0003] Although the existing screening systems have improved in throughput, they still have significant technical defects. Take the ER stress signal system as an example: although the monitoring mechanism based on endoplasmic reticulum membrane proteins such as Sec63 can evaluate the efficiency of protein folding, it cannot track the subsequent secretion steps (such as Golgi processing or vesicle transport), resulting in insufficient correlation between the signal and the extracellular secretion level (R²<0.6). In addition, ER stress is susceptible to cytotoxic interference (such as continuous activation triggering cell apoptosis), non-specific factors (nutritional stress, etc.) and dynamic range limitations (signal nonlinear saturation), resulting in a false positive rate of up to 30%-50%. On the other hand, microfluidic sorting technology relies on customized chips and precision fluid equipment. Some systems require the additional introduction of substrate activation reporter signals, and the cost of a single operation exceeds US$5,000, which seriously restricts the large-scale application of small and medium-sized laboratories. Summary of the invention

[0004] The present invention overcomes the above technical defects and provides a Pichia pastoris high secretion strain screening system and its use method. The present invention realizes efficient screening of secretory target proteins (such as plastic hydrolase LCC-ICCG) through the integration of bicistronic design, P2A peptide self-cleavage and cell surface display technology.

[0005] The technical solution of the present invention is as follows.

[0006] A Pichia pastoris high secretion strain screening system, the system uses Pichia pastoris as a host, the core structure of the system is as shown in the attachedFigure 1 As shown, the expression frame of the system includes the following elements: a promoter, a signal peptide, a target protein, a P2A peptide, a flexible connecting peptide, a reporter protein, an anchor protein and a terminator; the structure of the expression frame is: promoter-signal peptide-target protein-P2A peptide-signal peptide-reporter protein-flexible connecting peptide-anchor protein-terminator; the target protein is secreted through the signal peptide; the anchor protein displays the reporter protein on the surface of Pichia pastoris cells, and the fluorescent signal of the reporter protein displayed on the surface of Pichia pastoris cells represents the secretion level of the target protein.

[0007] Furthermore, the promoter is the methanol-inducible promoter AOX1; the terminator is the AOX1 terminator; and the signal peptide is the α-factor signal peptide.

[0008] like Figure 1 As shown, the core structure of the system of the present invention is: AOX1 promoter-α-factor signal peptide-target protein-P2A peptide-α-factor signal peptide-Gamillus-G4S connecting peptide-Gcw21 anchor sequence-AOX1 terminator. The corresponding full-length sequence is SEQ ID NO:1.

[0009] It should be noted that: The role of the AOX1 promoter is to drive the entire dual protein expression system, that is, to drive the co-expression of the target protein and the Gamillus fluorescent protein at the same time, and its role is to achieve the expression of two proteins in a single mRNA; the role of the AOX1 terminator is to ensure that RNA polymerase terminates transcription at the correct position to prevent transcription extension; the role of the double α-factor signal peptide is to be located at the N-terminus of the target protein (such as LCC-ICCG) and the Gamillus-Gcw21 complex, respectively, to guide the two into the secretory pathway. The target protein is secreted to the extracellular space through the signal peptide, while the Gamillus-Gcw21 complex is guided and finally anchored to the cell wall; the role of the P2A peptide is to connect the target protein and Gamillus, and promote ribosome jumping during the translation process, thereby achieving equimolar expression of two proteins on a single mRNA, that is, equimolar expression of two proteins; the role of the G4S flexible connecting peptide is to act as a flexible connector to enhance the structural freedom between Gamillus and Gcw21, reduce steric hindrance, ensure that each is independently and correctly folded, and improve the display efficiency.

[0010] Furthermore, the anchor protein is derived from the C-terminal GPI anchor domain of the Pichia pastoris cell wall protein Gcw21, which is used to fix the reporter protein to the outer layer of the cell wall. Its sequence corresponds to GenBank accession number: XP_002491452.1. The anchor protein is Gcw21, and the amino acid sequence of Gcw21 is SEQ ID NO:7.

[0011] Furthermore, high-secreting strains were screened by any of the following methods: (1) flow cytometry to sort bacterial colonies with high fluorescence signal intensity; (2) droplet microfluidics to separate individuals with single-cell fluorescence signal values ​​≥ threshold Z; (3) deep-well plate high-throughput culture combined with microplate reader fluorescence intensity analysis to identify and select strains with high fluorescence intensity.

[0012] Furthermore, the amino acid sequence of the P2A peptide is derived from the 2A self-cleavage peptide of porcine teschovirus-1 (PTV-1), and the gene sequence is SEQ ID NO: 4; wherein the combination of the P2A peptide and the signal peptide realizes the co-secretion of the target protein and the reporter protein, and the correlation coefficient of the co-expression amount R 2 ≥0.9.

[0013] Furthermore, the target protein is a secretory heterologous recombinant protein suitable for a secretory expression system of Pichia pastoris. The target protein is a fluorescent protein or a hydrolase; the fluorescent protein is an mCherry fluorescent protein, whose sequence corresponds to the FPbase database ID: ZERB6, and whose amino acid sequence is: SEQ ID NO: 3; The reporter protein is a fluorescent protein, the fluorescent protein is a Gamillus fluorescent protein, its sequence corresponds to FPbase database ID: 21PQ5, and its amino acid sequence is SEQ ID NO: 5; the hydrolase is selected from lipase, plastic hydrolase or β-galactosidase; the plastic hydrolase is plastic hydrolase LCC-ICCG, and its amino acid sequence is SEQ ID NO: 2.

[0014] Furthermore, the flexible connecting peptide is located between the reporter protein and the anchor protein; preferably, the flexible connecting peptide is a G4S connecting peptide, and its amino acid sequence is: GGGGS.

[0015] A method for using a Pichia pastoris high-secreting strain screening system comprises the following steps: (1) Construction of vector: constructing the sequences encoding the target protein, P2A peptide, reporter protein and anchor protein into an expression vector; the expression vector comprises a methanol-inducible promoter and an α-factor signal peptide; (2) Yeast transformation: Transform the constructed vector into Pichia pastoris to obtain positive transformants; (3) Screening strains: Cultivate positive transformants under induction conditions, detect the fluorescence signal intensity by flow cytometry, and sort the 10% or 1% of the cell population with the highest signal; wherein the fluorescence signal intensity is positively correlated with the secretion level of the target protein.

[0016] The specific steps are: 1. Vector Construction a. A functional DNA fragment of the anchoring protein anchoring domain was amplified from the genome of Pichia pastoris GS115 strain using DNA polymerase; b. Using a homologous recombination kit, the functional DNA fragment of the anchoring protein anchoring domain was cloned into the pPICZα A plasmid to construct the pPICZα A-Gcw21 plasmid; c. Add the target protein, P2A peptide, second α-factor signal peptide, reporter protein, and flexible linker peptide sequence to the pPICZα A-Gcw21 plasmid to construct the final plasmid pJC2405A; d. Plasmid pJC2405A was digested with restriction endonuclease Bgl II, and the digestion product was recovered to obtain pJC2405A digestion recovery product; 2. Transformation and screening a. The pJC2405A digestion product obtained in step 1 was added to Pichia competent cells and transformed by electroporation; b. Screening the transformed strains, culturing and selecting monoclonal colonies; c. Lyse the monoclonal colonies and verify the correctness of the plasmid by PCR and sequencing; d. Inoculate the verified correct strain into the culture medium, shake the bacteria, wash the cells and resuspend them in BMMY medium; e. Methanol was added to a final concentration of 1%, fermentation was induced, and samples were taken for fluorescence detection screening to obtain bacterial colonies with high fluorescence reporting values; 3. Activation and selection of cloned strains after screening a. Activate and culture the selected bacterial colonies and pick monoclonal colonies; b. Dissolve the picked monoclonal colonies in NaOH solution, lyse them, and use the lysate as a template to perform PCR amplification using universal primers; c. Recover the PCR product for sequencing and pick out the monoclonal strain; 4. Fermentation, induced expression and product collection a. Inoculate the monoclonal strain into YPD medium, shake the bacteria, centrifuge, wash and resuspend the cells in BMMY medium; b. Methanol was added to the culture medium to a concentration of 1% to induce fermentation; c. After inducing fermentation, centrifuge and separate the supernatant and precipitate; wash the precipitate and centrifuge for fluorescence detection; d. Screen out strains that secrete target proteins at high yields based on fluorescence intensity data; e. Collect the products of the strains that secrete the target protein with high yield for subsequent experiments.

[0017] The invention is as follows: 1. Design of dual signal peptide-P2A synergistic secretion: This system uses a double α-factor signal peptide to guide the secretion of the target protein and anchor the reporter protein to the cell surface, and introduces the P2A peptide (sequence: ATNFSLLKQAGDVEENPGP) derived from PTV-1 to achieve equimolar co-expression of the target protein (such as LCC-ICCG) and the Gamillus fluorescent protein at the same mRNA level. Since the two maintain a 1:1 ratio during transcription and translation and undergo the same secretion pathway, the final fluorescent signal is highly correlated with the secretion amount of the target protein (R²≥0.9), significantly reducing the risk of false positive screening (see Figure 4 ). In contrast, the traditional ER stress signal screening system based on Sec63 endoplasmic reticulum membrane protein can reflect protein folding efficiency, but cannot track subsequent secretion steps (such as Golgi processing or vesicle transport), so its reporter signal has a low correlation with the extracellular secretion level. The system of the present invention shows higher consistency and credibility between the fluorescent signal and the secretion capacity.

[0018] 2. The present invention optimizes and selects the Gcw21 anchor protein, and the display efficiency is better than that of the traditional anchor protein; 3.Full process high throughput compatible: Optimizing reporter protein spatial orientation through G4S flexible linker peptide (GGGGS) supports single FACS sorting of >10 6 cell( Figure 3 ), which takes only 2 hours and is 200 times more efficient than traditional shake flask screening (3-4 weeks).

[0019] Compared with the prior art, the advantages of the present invention are: 1. The system uses dual signal peptides and P2A peptides to enable co-secretion of the target protein and reporter protein, and the fluorescence signal is highly correlated with the secretion level (R²≥0.9).

[0020] 2. The Pichia pastoris high-secreting strain screening system of the present invention has high-throughput compatibility and supports single-time screening of >10 by flow cytometry (FACS). 6 A clone.

[0021] 3. Low cost, universality and scalability: Compared with the screening system that can only rely on droplet microfluidics technology, the screening system of the present invention has higher compatibility. It supports flow cytometry, which has a high penetration rate and has lower equipment construction costs than droplet microfluidics systems. This system is also compatible with conventional deep-well plate culture, and the cost of single bacteria verification is lower than 0.1, significantly reducing the experimental cost. In addition, this system has good scalability, and the anchor protein can be applied to other fungi, breaking through the limitations of host selection in the existing technology and expanding its application range. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the arrangement of the genetic elements in the present invention.

[0023] Figure 2A After the recombinant Pichia pastoris strain X33-pJC2405A was cultured with methanol for 48 hours, the intracellular distribution of mCherry fluorescent protein was photographed under a fluorescence microscope using the RFP red light excitation wavelength; Figure 2B To use GFP green light excitation wavelength to photograph the distribution of Gamillus fluorescent protein on the cell surface of the recombinant Pichia pastoris strain X33-pJC2405A after methanol induction culture for 48 hours; Figure 2C for Figure 2A and Figure 2B Integration display situation diagram; Figure 2D This is a diagram showing the distribution of mCherry fluorescent eggs photographed with red light excitation wavelength and Gamillus fluorescent protein photographed with green light excitation wavelength under a laser confocal microscope.

[0024] Figure 3 The recombinant Pichia pastoris strain X33-pJC2405A was cultured with methanol for 48 hours, and the culture was washed with PBS (pH=7.4) buffer. The cells were suspended and loaded on a flow cytometer for analysis to obtain the expression of mCherry and Gamillus in each single cell.

[0025] Figure 4 This is the result of linear regression analysis of the correlation between the expression levels of mCherry and Gamillus in strains with two copies of the expression cassette under flow cytometry detection.

[0026] Figure 5 This is a statistical diagram of the secretion efficiency of the high secretion expression strains screened by the system of the present invention.

[0027] Figure 6 The enzyme activities of the LCC-ICCG samples screened and expressed by the system of the present invention were compared with those of the standard enzyme solution (LCCc) expressed by Escherichia coli in the pNPB reaction system. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques may be used. Reagents or instruments used without indicating the manufacturer are considered to be conventional products that can be purchased commercially.

[0029] The materials and equipment required in the embodiments are as follows: Table 1. Material information table

[0030] Table 2. Instrument information

[0031] The specific implementation steps are as follows: 1. Vector Construction a. Amplify the functional DNA fragment of the Gcw21 anchoring domain from the genome of Pichia pastoris GS115 using DNA polymerase. The primer sequences used are as follows: JC23019F: 5' -tggtggaggttctgcggccgaccagcgaatctctgtcacc- 3' (Seq_1) JC23028R: 5'-GCTGGGCCACGTGAATTCttataacaaagcagcggcggc-3' (Seq_2) b. Using a homologous recombination kit, the Gcw21 DNA fragment was cloned into the pPICZα A plasmid provided by GenScript to construct the pPICZα A-Gcw21 plasmid. The sequence of the Gcw21 DNA fragment is: SEQ ID NO: 7.

[0032] c. Add mCherry fluorescent protein, P2A peptide, Gamillus fluorescent protein, and G4S connecting peptide sequence to the pPICZαA-Gcw21 plasmid to construct the final plasmid pJC2405A, whose full-length sequence is SEQ ID NO: 1.

[0033] d. The plasmid pJC2405A was digested with restriction endonuclease Bgl II, and the digestion product was recovered using a DNA fragment recovery kit to obtain pJC2405A Bgl II digestion recovery product.

[0034] 2. Transformation and screening a. Add the pJC2405A Bgl II digestion product to Pichia pastoris X33 competent cells for electroporation transformation.

[0035] b. Use Zeocin resistance screening reagent to screen and pick monoclonal colonies.

[0036] c. Dissolve the selected monoclonal colonies in 10 μL of 0.1% NaOH solution and lyse at 98°C for 10 minutes.

[0037] d. Take 1 μL of lysate as a template and use a pair of universal primers 5AOX and 3AOX for PCR amplification to verify whether the expected double bands are obtained. The primer sequences are as follows: 5AOX: 5'-CTggTTCCAATTgACAAgC- 3' (Seq_3) 3AOX: 5'-TggCATTCTgACATCCTC- 3' (Seq_4) e. Recover the PCR product, perform gel electrophoresis and sequencing, verify that the plasmid is constructed correctly, and then pick out the monoclonal strain.

[0038] f. Inoculate the verified strain into YPD medium and measure OD after 48 hours of shaking. 600 The cells were washed and resuspended in BMMY medium.

[0039] g. Add methanol to a final concentration of 1%, start inducing fermentation and take samples for fluorescence detection.

[0040] 3. High-throughput screening (the three steps a, b, and c in this high-throughput screening can be used separately or in combination) a. Flow cytometer screening: After 48 hours of induction fermentation, dual-channel fluorescence detection (Ex / Em = 488 / 525nm, Ex / Em = 645 / 685nm) was performed using a flow cytometer. Based on the fluorescence intensity, the strains with the top 1% fluorescence intensity were screened for further cultivation.

[0041] b. Droplet microfluidic screening: After 48 hours of induction fermentation, the droplet microfluidic system combined with fluorescence detection (Ex / Em = 488 / 525nm, Ex / Em = 645 / 685nm) was used to separate individuals with single-cell fluorescence signal values ​​≥ threshold Z.

[0042] c. Deep-well plate high-throughput screening: After inducing single clones in deep-well plates for 48 hours, the fluorescence of the bacterial solution in each well was detected by an ELISA instrument to identify and screen the strains with the top 1% fluorescence intensity.

[0043] 4. Activation and clone picking after screening a. Activate and culture the cells sorted by flow cytometry. Inoculate the sorted cells onto YPD solid plate medium containing zeocin and culture at 30°C for 48 hours.

[0044] b. After confirming the cell activity, pick the colonies and perform colony PCR verification as in step 2. After the verification is correct, proceed to the next step.

[0045] 5. Fermentation and induced expression a. Inoculate the verified monoclonal strain into liquid YPD medium, incubate at 30 degrees Celsius for 48 hours, and measure the OD 600 value.

[0046] b. Centrifuge, wash, and resuspend the cells in BMMY medium to set the initial OD 600 = 1.0.

[0047] c. Add methanol to the culture medium to a final concentration of 1% to induce fermentation, and record the start time of induction (i.e., hour 0).

[0048] d. Samples were taken every 24 hours to detect the fluorescence intensity and monitor the cell growth curve.

[0049] 6. Final screening and product collection a. After 144 hours of induction, centrifuge the sample and separate the supernatant from the precipitate. Wash the precipitate three times with sterile water and repeat the centrifugation step.

[0050] b. Resuspend the pellet and supernatant, dilute 20-fold and add to a 96-well ELISA plate.

[0051] c. Use a microplate reader (Tecan Infinite 200) for fluorescence detection and set the wavelengths to: Gamillus (Ex = 490 nm, Em = 530 nm); mCherry (Ex = 560 nm, Em = 640 nm).

[0052] 7. Data analysis and result evaluation a. Record the intensity of each fluorescence detection and draw a graph of fluorescence intensity changes to analyze the relationship between expression level and induction time.

[0053] b. Based on the fluorescence intensity data, strains with high target protein production were screened for subsequent industrial production applications.

[0054] Example 1: System construction and verification (using mCherry as a model protein) mCherry fluorescent protein is a stable red fluorescent protein, which is widely used in research fields such as cell biology and molecular labeling. Its excellent fluorescence properties make it an ideal tool for labeling and analyzing protein, cell and molecular interactions. This embodiment adopts an expression system based on Pichia pastoris, and through an optimized screening method, an efficient production of mCherry protein is achieved, providing a convenient and scalable industrial production approach. This method has high efficiency, operability and low cost, and is suitable for large-scale production. In this embodiment, the sequence of mCherry fluorescent protein is SEQ ID NO:3.

[0055] 1. Vector Construction a. Extract DNA from the genome of Pichia pastoris GS115 strain using DNA polymerase, and amplify the functional DNA fragment containing the Gcw21 anchoring domain using PCR. The primer sequences used in this step are as follows: JC23019F: 5' -tggtggaggttctgcggccgaccagcgaatctctgtcacc- 3' (Seq_1) JC23028R: 5'-GCTGGGCCACGTGAATTCttataacaaagcagcggcggc-3' (Seq_2) b. Use a homologous recombination kit to clone the Gcw21 fragment into the pPICZα A plasmid to construct the pPICZα A-Gcw21 plasmid.

[0056] c. The gene sequences of mCherry fluorescent protein, P2A peptide, α-factor signal peptide, Gamillus fluorescent protein and G4S connecting peptide were synthesized and inserted into the pPICZα A-Gcw21 plasmid by enzyme digestion to obtain the final plasmid pJC2405A. (The gene sequence is SEQ ID NO: 1) d. Use restriction endonuclease Bgl II to perform single enzyme digestion on plasmid pJC2405A, and use DNA recovery kit to recover the digestion product.

[0057] e. Verify the correctness of plasmid construction by PCR and sequencing to ensure the correct insertion of mCherry protein.

[0058] 2. Transformation and screening a. Add the Bgl II digestion product of pJC2405A to Pichia pastoris X33 competent cells and transform them by electroporation.

[0059] b. Use Zeocin resistance screening reagent to screen and culture the screened strains on YPD plates. Select monoclonal colonies and perform PCR and sequencing verification.

[0060] c. Inoculate the selected strain into YPD medium, shake the culture for 48 hours, and measure the OD 600 value, and wash the cells with sterile water.

[0061] d. Resuspend the cells in BMMY medium and set the initial OD 600 = 1.0, and methanol was added to a final concentration of 1% to induce expression.

[0062] 3. Flow cytometry screening a. After 48 hours of induction, dual-channel fluorescence detection was performed using a flow cytometer with the wavelengths set to: Ex / Em = 488 / 525nm (Gamillus fluorescent protein) and Ex / Em = 645 / 685nm (mCherry).

[0063] b. Based on the fluorescence intensity, the top 10% of strains were selected for further sorting.

[0064] c. Use flow cytometry to sort the top 1% of cells with the strongest fluorescent signal intensity, and collect the sorted cells for plate activation culture.

[0065] 4. Post-screening activation and clone picking a. Inoculate the sorted cells into YPD solid medium and culture at 30°C for 48 hours to confirm cell activity.

[0066] b. Select single clones for NaOH lysis treatment and verify the integration of the target gene by PCR.

[0067] c. Analyze the recovered PCR products by PCR and gel electrophoresis to ensure the correctness of the target gene. Finally, select the monoclonal strain.

[0068] 5. Fermentation and induced expression a. Inoculate the verified monoclonal strain into YPD medium, shake the culture for 48 hours, and measure the OD 600 value.

[0069] b. After centrifugation and washing, resuspend the cells in BMMY medium in a deep-well plate and set the initial OD 600 = 1.0, and methanol was added to a final concentration of 1% to induce fermentation.

[0070] c. The start time of induced fermentation was recorded as 0 hour, and samples were taken every 24 hours for fluorescence intensity detection and cell growth curve monitoring.

[0071] 6. Final screening and product collection a. After 144 hours of deep-well plate induction, centrifuge the sample and separate the supernatant from the precipitate. Wash the precipitate three times with sterile water and repeat the centrifugation step.

[0072] b. Dilute the precipitate and supernatant 20 times respectively and add them to a 96-well ELISA plate. Use an ELISA reader for fluorescence detection with the wavelengths set to: Gamillus (Ex = 490 nm, Em = 530 nm); mCherry (Ex = 560 nm, Em = 640 nm).

[0073] c. Record the fluorescence intensity data, screen out strains that secrete mCherry protein with high yield, and use them for subsequent large-scale production.

[0074] 7. Data Analysis and Results Evaluation a. Record the intensity of each fluorescence detection, draw a graph of fluorescence intensity changes, and analyze the relationship between expression level and induction time.

[0075] b. Analyze the ratio of the mCherry fluorescence intensity in the culture supernatant of each sample at each time point to the total mCherry fluorescence intensity inside and outside the cell to calculate the secretion efficiency of the target protein.

[0076] c. Based on the fluorescence intensity data, strains with high mCherry protein production were screened and further applied to industrial production to evaluate their performance in large-scale fermentation.

[0077] The results are as follows Figure 3 and Figure 4 As shown in Figure 2, the expression levels of the target protein and reporter protein in this system are highly correlated (R 2 >0.9) The screening effect is obvious, Figure 3 In the figure, A is the copy of expression cassette 1; B is the copy of expression cassette 2. Figure 5 It can be seen that the secretion efficiency of the target protein screened by this system is as high as 85%.

[0078] Example 2: Screening and obtaining a Pichia pastoris strain that secretes high-yield plastic hydrolase (using LCC-ICCG as a model protein) using the system of the present invention LCC-ICCG is an efficient plastic hydrolase that can hydrolyze polyester materials such as polyethylene terephthalate (PET) and has wide application value in the fields of biodegradable plastics and environmental remediation. In this example, a high-yield LCC-ICCG strain was screened using an expression system based on Pichia pastoris, and its degradation activity was determined to promote its application in the environmental protection industry. In this example, the amino acid sequence of LCC-ICCG is SEQ ID NO: 2.

[0079] 1. Vector construction and transformation a. The LCC-ICCG gene was constructed using whole gene synthesis technology and optimized in Pichia pastoris to make the codon adaptation index (CAI) > 0.8 to improve the expression efficiency in Pichia pastoris.

[0080] b. Using a homologous recombination kit, the LCC-ICCG gene was inserted into the screening system vector pJC2405A of the present invention as the target protein, replacing the mCherry fluorescent protein, and transformed into Pichia pastoris X33 competent cells.

[0081] c. Screen positive clones using Zeocin resistance screening reagent, verify correct gene integration using PCR, and select the correct monoclonal strain.

[0082] 2. Initial fermentation and induced expression a. Inoculate the verified monoclonal strain into YPD medium, culture at 30°C for 48 hours, and measure the OD 600 value.

[0083] b. Collect the cells by centrifugation, wash them three times with sterile water, and resuspend them in BMMY medium. The starting OD 600 Set to 1.0.

[0084] c. Add methanol to a final concentration of 1% and induce fermentation at 30°C and 250 rpm. Add methanol every 24 hours to maintain a final concentration of 1%. Take samples regularly to analyze the fluorescence intensity and cell growth curve.

[0085] 3. Screening of high secretion expression strains a. After 48 hours of induction, single-channel fluorescence detection was performed using flow cytometry (Ex / Em = 488 / 525nm).

[0086] b. Select the top 1% of the strains with the highest fluorescence signals for further activation and screening.

[0087] c. Referring to Example 1, a high-expression, high-activity LCC-ICCG expression strain was selected for large-scale fermentation optimization.

[0088] 4. Final fermentation and product collection After inducing fermentation for 144 hours according to step 2 above, the fermentation broth was centrifuged at 4000 rpm for 10 min to separate the supernatant and bacterial cells, and the supernatant was used as the crude enzyme solution for enzyme activity determination.

[0089] 5. LCC-ICCG enzyme activity assay The activity of LCC-ICCG was determined using ethyl p-nitrobenzoate (pNPB) as a substrate. The specific steps are as follows: a. Prepare 100 mM pNPB stock solution (dissolved in methanol) and store in the dark.

[0090] b. In a 96-well plate, add 188 μL of PBS buffer (100 mM, pH 8.0) to each well.

[0091] c. Take 10 μL of crude enzyme solution samples of different dilution multiples (such as 1 / 5, 1 / 2, original solution) and add them to the reaction wells. Use 10 μL of LCC-ICCG protein expressed and purified by E. coli (concentrations 20 nM, 40 nM, 80 nM) as the standard reference.

[0092] d. Add 2 μL of 100 mM pNPB to make the final reaction system substrate concentration 1 mM.

[0093] e. Detect the absorbance at 405 nm (OD 405 ), read once every minute for a total of 10 minutes.

[0094] 6. Enzyme activity calculation and result analysis a. Calculate OD 405 The relative enzyme activity of LCC-ICCG was determined by the rate of change of absorbance and compared with that of the standard.

[0095] b. Calculation of unit enzyme activity (U): 1 U is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol from the substrate per minute at 37°C and pH 8.0.

[0096] c. The results are as follows Figure 6 As shown, the data indicate that in a 200 μL pNPB reaction system, the enzyme activity of the LCC-ICCG crude enzyme solution (8 μL) is at least equivalent to that of the 40 nM LCCc standard enzyme solution expressed by Escherichia coli, that is, the high-yield LCC-ICCG red yeast strain screened by the system of the present invention has a crude enzyme activity that is at least equivalent to that of the 40 nM purified standard enzyme solution, showing high enzyme activity and excellent expression level, and has good industrial application prospects.

[0097] Example 3: Final screening of Pichia pastoris strains that secrete high-yield lipolysis enzymes (using Candida antarctica lipase B, CALB as a model protein) using the system of the present invention, product collection, and enzyme activity detection Lipase B (CALB) is an important industrial enzyme widely used in the fields of food, pharmaceuticals, and chemicals. It has the advantages of a wide range of catalytic substrates, high selectivity, and good stability. It has a huge market demand and high economic value. This example constructs an expression and high-throughput screening system based on Pichia pastoris to achieve efficient expression of CALB and rapid screening of high-yield strains, and detects and evaluates the activity of the strain expression product to clarify its application potential.

[0098] 1. Vector construction and transformation a. The lipase B (CALB) gene was constructed using whole gene synthesis technology: the amino acid sequence was SEQ ID NO: 8, and the Pichia pastoris was optimized to make the codon adaptation index (CAI) > 0.8 to improve the expression efficiency in Pichia pastoris.

[0099] b. Using a homologous recombination kit, the lipase B (CALB) gene was inserted into the screening system vector pJC2405A of the present invention as the target protein, replacing the mCherry fluorescent protein, and transformed into Pichia pastoris X33 competent cells.

[0100] c. Screen positive clones using Zeocin resistance screening reagent, verify correct gene integration using PCR, and select the correct monoclonal strain.

[0101] 2. Initial fermentation and induced expression a. Inoculate the verified monoclonal strain into YPD medium, culture at 30°C for 48 hours, and measure the OD 600 value.

[0102] b. Collect the cells by centrifugation, wash them three times with sterile water, and resuspend them in BMMY medium. The starting OD 600 Set to 1.0.

[0103] c. Add methanol to a final concentration of 1% and induce fermentation at 30°C and 250 rpm. Add methanol every 24 hours to maintain a final concentration of 1%. Take samples regularly to analyze the fluorescence intensity and cell growth curve.

[0104] 3. Screening of high secretion expression strains a. After 48 hours of induction, single-channel fluorescence detection was performed using flow cytometry (Ex / Em = 488 / 525nm).

[0105] b. Select the top 1% of strains with the highest fluorescence signals for PCR verification and further screen them through crude enzyme activity test.

[0106] c. Referring to Example 1, a high-expression, high-activity CALB expression strain was selected for large-scale fermentation optimization.

[0107] 4. Final fermentation and product collection After inducing fermentation for 144 hours according to step 2 above, the fermentation broth was centrifuged at 4000 rpm for 10 min to separate the supernatant and bacterial cells, and the supernatant was used as the crude enzyme solution for enzyme activity determination.

[0108] 5. CALB enzyme activity assay The CALB activity was determined using ethyl p-nitrobenzoate (pNPB) as a substrate. The specific steps are as follows: a. Prepare 100 mM pNPB stock solution (dissolved in methanol) and store in the dark.

[0109] b. In a 96-well plate, add 188 μL of PBS buffer (100 mM, pH 8.0) to each well.

[0110] c. Take 10 μL of crude enzyme solution samples of different dilution multiples (such as 1 / 5, 1 / 2, original solution) and add them to the reaction wells. Use 10 μL of purchased purified CALB protein (concentrations 20 nM, 40 nM, 80 nM) as a standard reference.

[0111] d. Add 2 μL of 100 mM pNPB to make the final reaction system substrate concentration 1 mM.

[0112] e. Detect the absorbance at 405 nm (OD 405 ), read once every minute for a total of 10 minutes.

[0113] 6. Enzyme activity calculation and result analysis a. Calculate OD 405 The relative enzyme activity of CALB was determined by the rate of change of absorbance and compared with that of the standard.

[0114] b. Calculation of unit enzyme activity (U): 1 U is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol from the substrate per minute at 37°C and pH 7.5.

[0115] Example 4: Screening and obtaining high-yield β-galactosidase secretion strains (using LacZ as a model protein) using the system of the present invention Pichia pastoris strains β-galactosidase (LacZ) is a commonly used hydrolase, which is widely used in research fields such as molecular biology, gene expression analysis, and biomarkers. Its enzyme activity level determines the sensitivity of detection methods such as X-gal color development and ONPG hydrolysis, and is crucial for screening recombinant strains with high expression. In this example, a high-yield LacZ strain was screened through a Pichia pastoris expression system, and its enzyme activity was determined to optimize its industrial production and scientific research application potential.

[0116] 1. Vector construction and transformation a. The LacZ gene was optimized for Pichia pastoris to make the codon adaptation index (CAI)>0.8 to improve the expression efficiency in Pichia pastoris. The LacZ gene was inserted into the screening system vector pJC2405A of the present invention as the target protein using a homologous recombination kit, replacing the mCherry fluorescent protein, and transformed into Pichia pastoris X33 competent cells.

[0117] The amino acid sequence of LacZ is SEQ ID NO:9 b. Screen positive clones using Zeocin resistance screening reagent, verify correct gene integration using PCR, and select correct transformants.

[0118] 2. Initial fermentation and induced expression a. Inoculate the verified monoclonal strain into YPD medium, culture at 30°C for 48 hours, and measure the OD 600 value.

[0119] b. Collect the cells by centrifugation, wash them three times with sterile water, and resuspend them in BMMY medium. The starting OD 600 Set to 1.0.

[0120] c. Add methanol to a final concentration of 1% and induce fermentation at 30°C and 250 rpm. Add methanol every 24 hours to maintain a final concentration of 1%. Take samples regularly to analyze the fluorescence intensity and cell growth curve.

[0121] 3. Screening of high secretion expression strains according to Example 1 a. After 48 hours of induction, single-channel fluorescence detection was performed using flow cytometry (Ex / Em = 488 / 525nm).

[0122] b. Select the top 1% of strains with the highest fluorescence signals for PCR verification and further screen them through crude enzyme activity test.

[0123] c. Select LacZ expression strains with high secretion expression and high activity for large-scale fermentation optimization.

[0124] 4. Final fermentation and product collection After inducing fermentation for 144 hours according to step 2 above, the fermentation broth was centrifuged at 4000 rpm for 10 min to separate the supernatant and bacterial cells, and the supernatant was used as the crude enzyme solution for enzyme activity determination.

[0125] 5. β-galactosidase activity was determined using ONPG (o-nitrophenyl-β-D-pyranogalactoside).

[0126] a. Preparation of reaction system ONPG stock solution: Prepare 10 mg / mL ONPG solution and store at -20°C in the dark. Add 190 μL PBS (50 mM, pH 7.4) to each well of a 96-well plate. Take 10 μL of the strain suspension at different dilutions and add it to the reaction wells, and add standard LacZ protein (10 nM, 20 nM, 50 nM, 100 nM) as a control.

[0127] b. Enzymatic reaction and detection Add 2 μL ONPG substrate to a final concentration of 0.1 mg / mL. Incubate at 37°C for 10 min and monitor the absorbance at 420 nm (OD 420 OD was recorded every 1 min. 420 , and continued monitoring for 10 min.

[0128] c. Enzyme activity calculation and screening Calculate OD 420 The β-galactosidase activity was determined by the rate of change of absorbance and compared with that of the standard.

[0129] Definition of enzyme activity unit (U): 1 U = the amount of enzyme required to hydrolyze ONPG to produce 1 μmol of o-nitrophenol (ONP) per minute at 37°C and pH 7.4.

[0130] 6. X-gal color verification The X-gal colorimetric assay is based on the hydrolysis of 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal) by LacZ to generate a blue insoluble product, which is used to quickly screen high-expressing strains.

[0131] a. Preparation of X-gal colorimetric plates X-gal mother solution: weigh 20 mg X-gal, dissolve in 1 mL DMF (anhydrous dimethylformamide), store at -20℃ away from light for later use. Color development plate: add X-gal (final concentration 40 μg / mL) to YPD medium, autoclave and pour the plate at below 50℃, store at 4℃ away from light.

[0132] b. Apply the Lacz expression strain screened by this system Take the high, medium and low expression Pichia yeast transformant bacterial liquid, spread 10 μL on the X-gal color plate, and culture at 30℃ for 2~4 days. Observe the color change of the colony: •Dark blue colonies: High expression of LacZ and high enzyme activity.

[0133] •Light blue colonies: low LacZ expression level.

[0134] •White colonies: no LacZ expression, negative clones.

[0135] Dark blue colonies were selected for subsequent quantitative enzyme activity determination.

[0136] 7. Analysis of screening results Combined with X-gal colorimetric screening and ONPG quantitative determination, strains with the highest enzyme activity and good stability were selected for subsequent large-scale fermentation and industrial application evaluation. The results showed that the high-expression LacZ strain obtained by the screening system of the present invention had a significantly higher β-galactosidase production than the ordinary expression strain, and was suitable for gene expression detection, biosensor development and industrial production.

Claims

1. A Pichia pastoris high secretion strain screening system, characterized in that: The system uses Pichia pastoris as a host; the system includes an expression cassette, which includes the following elements: a promoter, a signal peptide, a target protein, a P2A peptide, a flexible connecting peptide, a reporter protein, an anchor protein and a terminator; the structure of the expression cassette is: promoter-signal peptide-target protein-P2A peptide-signal peptide-reporter protein-flexible connecting peptide-anchor protein-terminator; the target protein is secreted through the signal peptide; the anchor protein displays the reporter protein on the surface of the Pichia pastoris cells, and the fluorescent signal of the reporter protein displayed on the surface of the Pichia pastoris cells represents the secretion level of the target protein.

2. The system according to claim 1, characterized in that High-secreting strains were obtained by sorting the 10% cell population with the highest fluorescence signal intensity using flow cytometry.

3. The system according to claim 1, characterized in that High-secreting strains were cultured in deep-well plates with high-throughput technology combined with a microplate reader for bacterial fluid fluorescence analysis, and the 1% cell population with the highest fluorescence signal intensity was selected.

4. The system according to claim 1, characterized in that The promoter is the methanol-inducible promoter AOX1; the terminator is the AOX1 terminator; and the signal peptide is the α-factor signal peptide.

5. The system according to claim 1, wherein: The amino acid sequence of the P2A peptide is SEQ ID NO: 4; the combination of the P2A peptide and the signal peptide achieves co-secretion of the target protein and the reporter protein, and the co-expression correlation coefficient R 2 ≥0.

9.

6. The system according to claim 1, wherein: The anchoring protein is the Gcw21 anchoring protein.

7. The system according to claim 1, characterized in that The target protein is a secreted heterologous recombinant protein.

8. The system of claim 1, wherein: The reporter protein is a fluorescent protein, and the fluorescent protein is a Gamillus fluorescent protein; the target protein is selected from any one of the following categories: (a) hydrolase: lipase, plastic hydrolase or β-galactosidase; or (b) fluorescent protein: mCherry fluorescent protein.

9. The system according to claim 1, characterized in that The flexible connecting peptide is a G4S connecting peptide.

10. A method for using the system according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Construction of vector: constructing the sequences encoding the target protein, P2A peptide, reporter protein and anchor protein into an expression vector; the expression vector comprises a methanol-inducible promoter and an α-factor signal peptide; (2) Yeast transformation: Transform the constructed vector into Pichia pastoris to obtain positive transformants; (3) Screening strains: Cultivate positive transformants under induction conditions, detect the fluorescence signal intensity by flow cytometry, and sort the 10% or 1% of the cell population with the highest signal; wherein the fluorescence signal intensity is positively correlated with the secretion level of the target protein.

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