Cell wall protein PpSAG1 and its application

Abstract

The present invention discloses a cell wall protein PpSAG1, whose amino acid sequence is shown in SEQ ID No. 2 and DNA sequence is shown in SEQ ID NO. 1. By using the cell wall protein PpSAG1 to express UGT76G1 on the surface of Pichia pastoris cells, a recombinant Pichia pastoris engineering strain with high rebaudioside A production can be obtained.

Description

Technical Field

[0001] The invention relates to a cell wall protein PpSAG1 and application thereof, belonging to the technical field of protein expression. Background Art

[0002] Rebaudioside A (RA) is a high-intensity natural sweetener with many excellent properties extracted from the leaves of Stevia rebaudiana, accounting for 2-4% of the dry leaf weight. Its sweetness is 150-300 times that of sucrose, and its calorific value is only 1 / 300 of that of sucrose. It has a pure taste without aftertaste and tastes closer to sucrose, making it easy for people to accept. High-purity rebaudioside A has great application value as a sweetener in the fields of food, beverages, brewing, medicine, daily chemical industry, etc. Therefore, it is of great significance to study and develop methods for preparing high-purity rebaudioside A. [1] . At present, the methods for preparing high-purity rebaudioside A products mainly focus on the following aspects: screening and cultivating stevia varieties with high rebaudioside A production, improving the separation and purification methods for extracting rebaudioside A, etc. These traditional methods have the disadvantages of complicated process, high production cost, and poor enzymatic effect. At present, there have been studies on the construction of engineered bacteria for synthesizing rebaudioside A for biological fermentation production of rebaudioside A, such as synthesizing rebaudioside A by co-expressing sucrose synthase (SUS) and UDP-glycosyltransferase (UDP-glycosyltransferase, UGT) in Pichia pastoris to form a whole-cell catalytic system; or combining the intracellular expression of sucrose synthase in Pichia pastoris and the surface display of UDP-glycosyltransferase to synthesize rebaudioside A, and improving the efficiency of Pichia pastoris whole-cell biocatalysis by improving the fermentation process. Although the use of Pichia pastoris surface display can increase the yield of rebaudioside A to a certain extent, no studies have evaluated the effects of different anchoring proteins on the cell surface display effect. [2] .

[0003] At present, yeast cell surface display has been widely used in various fields, such as improving protein production and thermal stability, cDNA library screening, etc. In addition to utilizing the great advantages of yeast itself in fermentation, yeast surface display can form whole-cell catalysts by fixing catalytically active enzymes on the outer surface of yeast cells in a covalent or non-covalent manner. While maintaining its high efficiency, specificity and mild enzyme catalytic reaction characteristics, the spatial positioning of surface-displayed enzymes makes them have many excellent characteristics compared with free enzymes, such as improved temperature stability, resistance to organic solvents, repeated use, continuous controllable operation and simple process. Yeast surface enzyme display technology is similar to traditional immobilized enzyme technology, but it can avoid protein purification and fixation operations, and has good prospects in the application of biological enzyme catalysis. Therefore, it is of great practical significance to construct a surface-displayed strain of UDP-glycosyltransferase for the conversion of rebaudioside A, which provides application prospects for the industrialization of efficient enzymatic catalytic production of rebaudioside A.

[0004] In recent years, Pichia pastoris has gradually become the most commonly used eukaryotic expression platform for heterologous proteins due to its advantages such as high growth density, moderate protein post-translational modification, strong protein secretion ability, and simple culture process. Pichia pastoris has been recognized as a safe and reliable (GRAS) strain in the U.S. market by the U.S. Food and Drug Administration (FDA) and is approved for use in the pharmaceutical and food industries. The anchor proteins reported for Pichia pastoris surface display systems mainly include Sed1p and Aga1p from Saccharomyces cerevisiae and GCW61 from Pichia pastoris [3], which have been successfully used in the Pichia pastoris display system. Although surface display has many advantages, there is still the problem of low protein display capacity. The properties of the anchor protein itself have a great influence on the activity of the displayed enzyme. The surface display efficiency is still the key to its use. Therefore, it is necessary to select a suitable anchor protein to express a specific protein, thereby increasing the amount of protein displayed by Pichia pastoris. Summary of the invention

[0005] The present invention discovered a cell wall protein PpSAG1, whose amino acid sequence is shown in SEQ ID No.2.

[0006] The invention also discloses the coding gene of the cell wall protein PpSAG1.

[0007] Preferably, its nucleotide sequence is shown as SEQ ID No.1.

[0008] The invention also discloses the application of the cell wall protein PpSAG1 in constructing a recombinant Pichia pastoris engineering strain.

[0009] Preferably, the recombinant Pichia pastoris engineered strain displays glycosyltransferase UGT76G1 on its surface.

[0010] The present invention also discloses a surface display expression vector pPIC9K-UGT76G1-PpSAG1, wherein the coding gene of glycosyltransferase UGT76G1 and the coding gene of cell wall protein PpSAG1 are connected behind the promoter of pPIC9K vector, so as to express glycosyltransferase UGT76G1 and cell wall protein PpSAG1.

[0011] Generally speaking, the surface display expression vector includes a promoter, a 5' non-coding region, an α-factor secretory peptide, a UGT76G1 encoding gene, an anchor protein encoding gene and a 3' non-coding region, wherein the promoter is a constitutive promoter AOX1.

[0012] The expression vector may also include a selectable marker gene for screening transformed strains, including resistance genes encoding antibiotic tolerance and auxotrophic genes.

[0013] The invention also discloses a recombinant Pichia pastoris engineering strain, into which the surface display expression vector pPIC9K-UGT76G1-PpSAG1 is transformed.

[0014] Preferably, the Pichia pastoris is Pichia pastoris GS115.

[0015] The invention also discloses the application of the above-mentioned recombinant Pichia pastoris engineering strain in the preparation of rebaudioside A.

[0016] The invention provides a cell wall protein PpSAG1. The cell wall protein PpSAG1 is used to express UGT76G1 on the surface of Pichia pastoris cells, so as to obtain a recombinant Pichia pastoris engineering strain with high rebaudioside A production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the plasmid map of pPIC9K-UGT76G1-GCW61.

[0018] Figure 2 This is the plasmid map of pPIC9K-UGT76G1-Sed1p.

[0019] Figure 3 This is the plasmid map of pPIC9K-UGT76G1-Aga1p.

[0020] Figure 4 This is the plasmid map of pPIC9K-UGT76G1-PpSAG1.

[0021] Figure 5 These are the PCR amplification bands of each recombinant Pichia pastoris engineering strain.

[0022] Figure 6Comparison of rebaudioside A production of each recombinant Pichia pastoris engineered strain.

[0023] Figure 7 Comparison of the stevioside conversion rates of various recombinant Pichia pastoris engineered strains.

[0024] Figure 8 These are cell photos of each recombinant Pichia pastoris engineered bacteria under a fluorescence microscope. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the field, and the extraction of plasmids, RNA acquisition, DNA fragment recovery, ligation and other operations are detailed in the instructions of the corresponding kits. For details on the preparation of culture media and the cultivation of yeast cells, please refer to Invitrogen TM The company’s operating manual “User Guide: Pichia Expression Kit” (Source: https: / / www.thermofisher.cn / order / catalog / product / K171001# / K171001).

[0028] Example 1: Construction of a recombinant Pichia pastoris UGT76G1 surface display engineered strain to identify a novel and highly efficient anchoring protein.

[0029] 1. Extract the yeast strain genome

[0030] (1) Use a sterilized toothpick to pick a yeast colony of appropriate size from the plate and inoculate it into a 5 mL YPD tube. Cultivate at 220 rpm and 30°C for 48 h.

[0031] (2) Take 1.5 mL of bacterial solution, centrifuge at 12000 rpm for 1 min, discard the supernatant, collect the bacteria, resuspend and wash with 1 mL of deionized water, centrifuge at 12000 rpm for 1 min, and remove the supernatant;

[0032] (3) Add 5 mg of acid-washed glass sand to the cells collected in the previous step, then add 200 μL of yeast lysis solution and 200 μL of phenol:chloroform:isoamyl alcohol (25:24:1) mixture, vortex on a vortexer for 2 min; then add 200 μL of 1×TE (Tris-EDTA) buffer and invert several times;

[0033] (4) Place the mixed solution in a centrifuge and centrifuge at 12,000 rpm for 10 min. Then, remove the supernatant after stratification and transfer it to a new EP tube. Add 2.5 volumes of pre-cooled anhydrous ethanol and 0.1 volumes of sodium acetate solution (pH 5.2) and mix well.

[0034] (5) The mixed product was placed at -20°C and allowed to settle naturally for 1 hour;

[0035] (6) The product after precipitation was taken out, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and then 1 mL of 70% ethanol was added and vortexed for 10 s;

[0036] (7) Repeat step 6, remove the supernatant, and dry the precipitate at room temperature until it becomes translucent;

[0037] (8) Add 200 μL 1× TE buffer to dissolve genomic DNA; determine the DNA concentration and store in a -20°C refrigerator.

[0038] 2. Obtain anchor protein encoding gene

[0039] The genome of Pichia pastoris was used as a template to amplify the GCW61 and PpSAG1 coding gene sequences. The genome of Saccharomyces cerevisiae was used as a template to amplify the Sed1p and Aga1p coding gene sequences. The codon-optimized UGT76G1 coding gene sequence was used as a template to amplify the UGT76G1 coding gene sequence. The above fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Novozyme).

[0040] 3. Obtain E. coli plasmid pPIC9K

[0041] Plasmid pPIC9K was extracted using a plasmid extraction kit (Novagen).

[0042] 4. Get the linearized pPIC9K

[0043] pPIC9K was digested with endonucleases EcoRI and NotI at 37°C for 2 h. The fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Novozymes).

[0044] 5. Assembly of pPIC9K-UGT76G1 vector (Table 1)

[0045] The above-mentioned fragment ends were assembled using a seamless cloning kit (Novozyme) and connected to form various vectors. The vector map is shown in Figure 1-4 As shown, the coding gene sequence of GCW61 is shown as SEQ ID No.3, the coding gene sequence of Sed1p is shown as SEQ ID No.4, and the coding gene sequence of Aga1p is shown as SEQ ID No.5.

[0046] Table 1 pPIC9K-UGT76G1 vector

[0047] serial number Carrier 1 pPIC9K-UGT76G1-GCW61 2 pPIC9K-UGT76G1-Sed1p 3 pPIC9K-UGT76G1-Aga1p 4 pPIC9K-UGT76G1-PpSAG1

[0048] 6. Linearized vector

[0049] Each vector was digested with endonuclease SalI at 37°C for 2 h. The fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Novozyme).

[0050] 7. Transform the linearized vector into recombinant Pichia pastoris GS115:

[0051] (1) Pick a fresh and robust yeast single colony from the plate, inoculate it into 50 mL of YPD liquid medium, and culture it overnight at 220 rpm and 30°C;

[0052] (2) Cultivate until OD 600 When the RPMI is 1-2, transfer the bacterial solution to a sterilized 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the bacterial cells;

[0053] (3) Add 30 mL of lithium acetate sorbitol Tris mixed solution (LST buffer) to the collected bacteria for resuspending and let stand at 30°C for 30 min; centrifuge the bacterial solution at 5000 rpm for 5 min and remove the supernatant;

[0054] (4) The precipitated cells were resuspended in 10 mL of pre-cooled sorbitol solution, centrifuged at 5000 rpm for 5 min, and the supernatant was discarded; step (4) was repeated twice;

[0055] (5) Add about 400 μL of pre-cooled sorbitol solution to the bacteria collected for the last time, carefully resuspend, complete the preparation of Pichia pastoris competent cells, and dispense into sterilized pre-cooled EP tubes at 80 μL / tube for later use (make and use immediately);

[0056] (6) Add 5 μg of linearized plasmid (total volume not exceeding 20 μl) to 80 μl of Pichia competent medium, gently blow evenly with a gun, and incubate on ice for 5 min;

[0057] (7) Add the competent cells after mixing the plasmid into the pre-cooled electrode cup (0.1 cm spacing); set the voltage of the electroporator to 780 V, place the electrode cup into the electroporator, and prepare 1 mL of pre-cooled sorbitol solution in advance;

[0058] (8) Click the start button of the electroporator and record the voltage and shock time;

[0059] (9) Take out the electrode cup, add 1 mL of pre-cooled sorbitol solution prepared in advance, and mix well by pipetting;

[0060] (10) Place the mixed bacterial solution in a 30°C incubator and let it stand for 1.5 hours; spread the bacterial solution on an MD plate and place it in a 30°C incubator for 2-3 days. After a single clone grows, perform genotyping and screen for positive transformants. Figure 5 As shown (in the figure: M is DL5000 DNA Marker; Figure 5 - Lanes 1-8 in A are PCR amplification bands using the genome of the recombinant bacterium GS115::pPIC9K-UGT76G1-GCW61 as a template; Figure 5 - Lanes 1-8 in B are PCR amplification bands using the genome of the recombinant bacterium GS115::pPIC9K-UGT76G1-Sed1p as a template; Figure 5 - Lanes 1-8 in C are PCR amplification bands using the genome of the recombinant bacterium GS115::pPIC9K-UGT76G1-Aga1p as a template; Figure 5 - Lanes 1-8 in D are PCR amplification bands using the genome of the recombinant bacterium GS115::pPIC9K-UGT76G1-PpSAG1 as a template; ultimately, various recombinant Pichia pastoris engineered strains displaying UGT76G1 on the surface were obtained.

[0061] 8. Preparation of whole cell catalyst and catalytic reaction:

[0062] (1) The above recombinant Pichia yeast was selected and inoculated into BMGY medium for primary culture until OD 600 When the value is close to 5.0-6.0, collect the cells by centrifugation;

[0063] (2) Then resuspend the collected cells in BMMY medium to the starting OD 600 Close to 0.5-1.0, secondary culture was carried out at 30°C, methanol with a final concentration of 1% was added every day during the secondary culture, and the cells were recovered by centrifugation after 5 days of fermentation;

[0064] (3) After the bacterial cells are freeze-dried, a whole-cell catalyst containing the recombinant Pichia pastoris engineered bacteria is obtained.

[0065] (4) Prepare the reaction mixture according to the following components and their concentrations: 50 mM PBS buffer, 3 mM MgCl 2 , 250mM sucrose, 0.5-1mM UDPG, 3mg / mL steviol glycosides;

[0066] (5) adding 50 OD of whole-cell catalyst containing recombinant Pichia pastoris to the reaction system, reacting at 40° C. for 24 h and then terminating the reaction;

[0067] (6) The reaction solution was diluted 5 times, centrifuged and the supernatant was filtered and used for HPLC analysis;

[0068] (7) Detect the production of rebaudioside A and the conversion rate of stevioside in each recombinant Pichia pastoris engineered strain.

[0069] like Figure 6 and Figure 7 As shown, with the wild type GS115 as the control, the recombinant Pichia pastoris using PpSAG1 as the anchor protein had the highest yield of rebaudioside A and the highest conversion rate of stevioside.

[0070] 9. Immunofluorescence microscopy to verify surface display effects

[0071] (1) After methanol induction, take 800 μL of bacterial solution into a clean 2 mL centrifuge tube and control the OD 600 = 5 to 6. Centrifuge at 6000 rpm for 5 min at room temperature, discard the supernatant, and collect the yeast cells.

[0072] (2) Wash the cells three times with 1 mL of PBS solution (pH 7.4). After washing, centrifuge at 6000 rpm for 5 min, discard the supernatant, and collect the yeast cells.

[0073] (3) Resuspend the yeast cells with 800 μL of PBS solution (pH 7.4) containing 1% BSA and let stand for 10 min.

[0074] (4) Take 200 μL of yeast cell suspension, add 1 μL of mouse monoclonal antibody Anti-FLAG (2 μg / μL), and incubate in a 37°C constant temperature shaker at 60 rpm in the dark for 2 h.

[0075] (5) After incubation, centrifuge at 6000 rpm for 5 min, discard the supernatant, and collect the yeast cells. Wash the cells three times with 1 mL of PBS solution (pH 7.4). After washing, centrifuge at 6000 rpm for 5 min, discard the supernatant, and collect the yeast cells.

[0076] (6) Resuspend the yeast cells with 1 mL of PBS solution (pH 7.4) containing 1% BSA, let stand for 10 min, centrifuge at 6000 rpm for 5 min at room temperature, discard the supernatant, and collect the yeast cells.

[0077] (7) Resuspend the yeast cells with 200 μL of PBS solution (pH 7.4) containing 1% BSA, add 1 μL of AlexaFluor488-labeled goat anti-mouse IgG antibody (2 μg / μL), and incubate at 37°C in a constant temperature shaker at 60 rpm in the dark for 1 h.

[0078] (8) Wash the cells three times with 1 mL of PBS solution (pH 7.4). After washing, centrifuge at 6000 rpm for 5 min, discard the supernatant, and collect the yeast cells.

[0079] (9) Resuspend the yeast cells in 1 mL of PBS solution (pH 7.4) and observe under an immunofluorescence microscope.

[0080] The recombinant Pichia pastoris was observed under an immunofluorescence microscope (NIKONECLIPSE Ni-U) according to the method described in Example 1. Figure 8 As shown, green fluorescence can be observed on the cell surface of the recombinant Pichia pastoris engineered strain, indicating that UGT76G1 was successfully displayed on the cell surface.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0082] References:

[0083] [1] BASHARAT S, HUANG Z, GONG M, et al. A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana [J]. Chinese Journal of Chemical Engineering, 2021, 30: 92-104.

[0084] [2]WANG D,CHEN M,ZENG X,et al.Improving the catalytic performance ofPichia pastoris whole-cell biocatalysts by fermentation process[J].RSCAdvances,2021,11(57):36329-39.

[0085] [3]TEYMENNET-RAMíREZ K V,MARTíNEZ-MORALES F,TREJO-HERNáNDEZ M R.YeastSurface Display System:Strategies for Improvement and BiotechnologicalApplications[J].Frontiers in Bioengineering and Biotechnology,2022,9.

Claims

1. Application of cell wall protein PpSAG1 in constructing a recombinant Pichia pastoris engineering strain, characterized in that The amino acid sequence of the cell wall protein PpSAG1 is shown in SEQ ID No. 2, and the recombinant Pichia pastoris engineered strain displays glycosyltransferase UGT76G1 on its surface.

2. The use according to claim 1, characterized in that The coding gene sequence of the cell wall protein PpSAG1 is shown in SEQ ID No.

1.

3. A surface display expression vector pPIC9K- UGT76G1-PpSAG1 , characterized in that The promoter of the pPIC9K vector is connected to the gene encoding the glycosyltransferase UGT76G1 and the gene encoding the cell wall protein PpSAG1, wherein the amino acid sequence of the cell wall protein PpSAG1 is shown in SEQ ID No.

2.

4. The surface display expression vector pPIC9K- UGT76G1-PpSAG1, Features The coding gene sequence of the cell wall protein PpSAG1 is shown in SEQ ID No.

1.

5. A recombinant Pichia pastoris engineered strain, characterized in that The surface display expression vector pPIC9K- UGT76G1-PpSAG1 .

6. The recombinant Pichia pastoris engineered strain according to claim 5, characterized in that The Pichia pastoris is Pichia pastoris GS115.

7. Use of the recombinant Pichia pastoris engineered strain according to claim 5 or 6 in the preparation of rebaudioside A.

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