Method for improving cell surface display efficiency and product

Abstract

The invention discloses a method for improving cell surface display efficiency and a product, and belongs to the technical field of biology. The invention provides a method for improving cell surface display efficiency. The method comprises the following steps: S1, increasing the size of a cell; knocking out genes in the cells to increase the size of the cells; the gene is selected from a group consisting of a PASchr < 2-10508 >, a PASchr < 1-40585 >, a PASchr < 30819 >, a PASchr < 2-20036 >, a PASchr < 2-10366 >, a PASchr < 1-40244 >, a PASchr < 30688 >, a PASchr < 1-10383 >, a PASchr < 1-40081 > and a PASchr < 40223 >. According to the method for improving the cell surface display efficiency, ten single-gene knockout strains and ten double-gene knockout strains are obtained based on the method, and experiments prove that the cell sizes of the strains are increased to different extents, so that the display efficiency, the display quantity and the enzyme activity are improved in the aspect of cell surface display, and the cell surface display efficiency is improved. Meanwhile, the report protein UGT73F24m-Ag alpha of the strains catalyzes GA to obtain a GLMG product, and the concentration of the GLMG product is increased to different degrees.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method and product for improving the efficiency of cell surface display. Background Art

[0002] Cell surface display refers to the process of expressing a specific protein on the cell surface using recombinant DNA technology. Cell size is an important physiological characteristic of all organisms, and it is crucial to study the impact of its changes on the functions of organisms. However, current research on cell size mainly focuses on cell physiology such as the cell cycle, cell growth rate, and division relationship, and the objects of concern are more animal cells, Saccharomyces cerevisiae, and Escherichia coli, etc. There are few studies focusing on Pichia pastoris, an unconventional and excellent host.

[0003] In addition, yeast surface display, as one of the major applications of Pichia pastoris, is of great significance in improving the stability of enzymes and realizing enzyme cascade reactions. However, its low display efficiency limits its further industrial application. Although there are currently many strategies to improve the display efficiency, more attention is still focused on improving the transcriptional and translational efficiency of the target enzyme, and the display efficiency of the target enzyme is still limited by the number of anchoring sites on the surface of the host cell. In terms of improving the secretion of foreign proteins, different strength promoters (such as GAP / AOX1, etc.) can be replaced, or saturation mutagenesis can be performed on the signal peptide sequence and multiple copies of the target gene can be made, etc. For the latter, researchers mainly start from the anchoring protein. Usually, different anchoring proteins have different activities even for the same enzyme being displayed. Therefore, screening different anchoring proteins is of great significance for improving the display efficiency. In addition, in addition to using a single anchoring protein for yeast surface display, using double or multiple anchoring proteins to display foreign enzymes can, to a certain extent, improve the utilization rate of yeast surface anchoring sites and thus improve the display efficiency. However, whether it is to improve the secretion of the target protein or to use different types and numbers of anchoring proteins, the improvement of the display efficiency often targets a certain protein, that is, when changing a target protein, all strategies may need to be re-optimized and do not have universality. Therefore, finding a strategy that is effective for most proteins to improve their display efficiency has an important impact on the development of yeast surface display technology. Therefore, there is an urgent need in this field to develop more methods and products with better effects in improving the display efficiency. Summary of the Invention

[0004] In order to solve the technical problem of poor improvement effect of cell surface display efficiency, the present invention provides a method and product for improving the efficiency of cell surface display.

[0005] The present invention adopts the following technical solutions:

[0006] A method for improving the efficiency of cell surface display, which comprises the following steps: S1. increasing the size of the cell.

[0007] Knock out the gene in the cell to increase the size of the cell; the gene is selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223.

[0008] Knock out PAS_chr2-1_0508, or PAS_chr3_0819, or PAS_chr2-2_0036, or PAS_chr2-1_0366, or PAS_chr1-4_0244, or PAS_chr3_0688, or PAS_chr1-1_0383, or PAS_chr1-4_0081, or PAS_chr4_0223 in the cell, or simultaneously knock out the two genes PAS_chr2-1_0508 and PAS_chr2-1_0366 in the cell, or the two genes PAS_chr2-1_0508 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0508 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0508 and PAS_chr4_0223, the two genes PAS_chr2-1_0366 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0366 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0366 and PAS_chr4_0223, the two genes PAS_chr1-1_0383 and PAS_chr1-4_0081, the two genes PAS_chr1-1_0383 and PAS_chr4_0223, the two genes PAS_chr1-4_0081 and PAS_chr4_0223.

[0009] The knockout is carried out by homologous recombination.

[0010] The cell is yeast;

[0011] Preferably, the yeast is Pichia pastoris;

[0012] Preferably, the Pichia pastoris is UGT-Agα strain.

[0013] A product for improving the efficiency of cell surface display, which comprises: cells with increased size.

[0014] The cells with increased size are cells with the gene knocked out; the gene is selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223.

[0015] The cells with increased size are selected from: cells with the gene PAS_chr2-1_0508 knocked out, or the gene PAS_chr3_0819, or the gene PAS_chr2-2_0036, or the gene PAS_chr2-1_0366, or the gene PAS_chr1-4_0244, or the gene PAS_chr3_0688, or the gene PAS_chr1-1_0383, or the gene PAS_chr1-4_0081, or the gene PAS_chr4_0223, or cells with the double genes PAS_chr2-1_0508 and PAS_chr2-1_0366 knocked out simultaneously, or the double genes PAS_chr2-1_0508 and PAS_chr1-1_0383, the double genes PAS_chr2-1_0508 and PAS_chr1-4_0081, the double genes PAS_chr2-1_0508 and PAS_chr4_0223, the double genes PAS_chr2-1_0366 and PAS_chr1-1_0383, the double genes PAS_chr2-1_0366 and PAS_chr1-4_0081, the double genes PAS_chr2-1_0366 and PAS_chr4_0223, the double genes PAS_chr1-1_0383 and PAS_chr1-4_0081, the double genes PAS_chr1-1_0383 and PAS_chr4_0223, the double genes PAS_chr1-4_0081 and PAS_chr4_0223.

[0016] The cells are yeast.

[0017] Preferably, the yeast is Pichia pastoris.

[0018] Preferably, the Pichia pastoris is the UGT-Agα strain.

[0019] The product for improving the efficiency of cell surface display is selected from: KO-1 strain with gene PAS_chr2-1_0508 knocked out, or KO-3 strain with gene PAS_chr3_0819 knocked out, or KO-4 strain with gene PAS_chr2-2_0036 knocked out, or KO-5 strain with gene PAS_chr2-1_0366 knocked out, or KO-6 strain with gene PAS_chr1-4_0244 knocked out, or KO-7 strain with gene PAS_chr3_0688 knocked out, or KO-8 strain with gene PAS_chr1-1_0383 knocked out, or KO-9 strain with gene PAS_chr1-4_0081 knocked out, or KO-10 strain of cells with gene PAS_chr4_0223 knocked out, or KO-1+KO-5 strain with both genes PAS_chr2-1_0508 and PAS_chr2-1_0366 knocked out, or KO-1+KO-8 strain with both genes PAS_chr2-1_0508 and PAS_chr1-1_0383 knocked out, KO-1+KO-9 strain with both genes PAS_chr2-1_0508 and PAS_chr1-4_0081 knocked out, KO-1+KO-10 strain with both genes PAS_chr2-1_0508 and PAS_chr4_0223 knocked out, KO-5+KO-8 strain with both genes PAS_chr2-1_0366 and PAS_chr1-1_0383 knocked out, KO-5+KO-9 strain with both genes PAS_chr2-1_0366 and PAS_chr1-4_0081 knocked out, KO-5+KO-10 strain with both genes PAS_chr2-1_0366 and PAS_chr4_0223 knocked out, KO-8+KO-9 strain with both genes PAS_chr1-1_0383 and PAS_chr1-4_0081 knocked out, KO-8+KO-10 strain with both genes PAS_chr1-1_0383 and PAS_chr4_0223 knocked out, KO-9+KO-10 strain with both genes PAS_chr1-4_0081 and PAS_chr4_0223 knocked out.

[0020] The beneficial effects of the present invention are as follows:

[0021] Yeast surface display is one of the methods to immobilize foreign proteins by fusing an anchor protein with the foreign protein and fixing the foreign protein on the yeast cell surface, thereby achieving the immobilization of the target protein. Among yeast surface display hosts, Pichia pastoris has been widely used in yeast surface display due to its high secretion level of foreign proteins. Although yeast surface display using Pichia pastoris as the chassis cell has many advantages in the immobilization of foreign proteins, there are still problems such as low display efficiency of the target protein, which limits its wide industrial application. Currently, in order to improve the yeast surface display efficiency, mainly through overexpression regulation strategies to improve the secretion efficiency of enzymes, while the surface display efficiency of enzymes is still affected by the number of anchor sites in yeast cells themselves. Therefore, the present invention starts from the transformation of the chassis bacterium, specifically using Pichia pastoris as the chassis cell, by mining genes related to the size of Pichia pastoris, using gene knockout methods to explore the influence of related genes on the size of Pichia pastoris, and then exploring the influence of changes in cell size on the activity and display amount of enzymes displayed on the surface of Pichia pastoris, and further constructing a Pichia pastoris chassis for efficiently displaying foreign proteins. The present invention is of great significance for the related development of the cell morphology of Pichia pastoris, and provides a new idea for improving the yeast surface display efficiency with Pichia pastoris as the host.

[0022] The present invention provides a method for improving the cell surface display efficiency, and 10 single-gene knockout strains and 10 double-gene knockout strains are obtained based on this method. Through experiments, it is verified that the cell sizes of these strains have increased to varying degrees, and thus the display efficiency, display amount, and enzyme activity have been improved in cell surface display. At the same time, the concentrations of the GLMG products catalyzed by the reporter protein UGT73F24m-Agα of these strains from GA have been increased to varying degrees. The present invention pioneerly starts from regulating the cell size of Pichia pastoris, changes the size of Pichia pastoris by means such as gene knockout, and further uses UGT73F24m as the reporter protein, applying the strains with cell size changes to improving the yeast surface display efficiency, thereby proposing a general strategy starting from the chassis host and effective for improving the display efficiency of various enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the PCR electrophoresis result diagram of 10 single-gene knockout strains in Experimental Example 1 of the present invention.

[0024] Figure 2 It is the result diagram of the significant difference analysis of the average diameters of 10 single-gene knockout strains and the wild strain UGT-Agα in Experimental Example 1 of the present invention.

[0025] Figure 3 It is the bar chart of the average cell surface area and average cell volume of 10 single-gene knockout strains in Experimental Example 1 of the present invention.

[0026] Figure 4 PCR electrophoresis result diagram of 10 double gene knockout strains in Experimental Example 1 of the present invention.

[0027] Figure 5 Bar chart of the significant difference analysis between 10 double gene knockout strains and their corresponding single gene knockout strains in Experimental Example 1 of the present invention.

[0028] Figure 6 Result diagram of the significant difference analysis between 10 double gene knockout strains and UGT-Agα in Experimental Example 1 of the present invention.

[0029] Figure 7 Bar chart of the average cell surface area and average cell volume of 10 double gene knockout strains in Experimental Example 1 of the present invention.

[0030] Figure 8 Bar chart of the measurement of the dry cell weight in Experimental Example 1 of the present invention.

[0031] Figure 9 Growth curve diagram of the double gene knockout strain in Experimental Example 1 of the present invention.

[0032] Figure 10 Schematic diagram of the principle of UGT73F24m-Agα catalyzing GA in Experimental Example 2 of the present invention.

[0033] Figure 11 Bar chart of the enzyme amount displayed by a single cell of the single gene knockout strain in Experimental Example 2 of the present invention.

[0034] Figure 12 Concentration of GLMG generated by a single cell of the single gene knockout strain in Experimental Example 2 of the present invention.

[0035] Figure 13 Bar chart of the comparison of cell diameter, display amount and activity of strains KO-1, KO-5, KO-8, KO-9, KO-10 in Experimental Example 2 of the present invention.

[0036] Figure 14 Bar chart of the enzyme amount displayed by a single cell of the double gene knockout strain in Experimental Example 2 of the present invention.

[0037] Figure 15 Bar chart of the concentration of GLMG generated by a single cell of the double gene knockout strain in Experimental Example 2 of the present invention.

[0038] Figure 16 Bar chart of the comparison of average diameter, display amount and activity of strains KO-9+KO-10 with KO-9, KO-10 and UGT-Agα in Experimental Example 2 of the present invention. Detailed implementation manner

[0039] The present invention will be further described in detail below in conjunction with specific embodiments and experimental examples, so that those skilled in the art can more clearly understand the present invention. The following experimental examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following experimental examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following experimental examples are all commercially available products well-known to those skilled in the art unless otherwise specified.

[0040] Sources of Biomaterials

[0041] The strain UGT-Agα used in Experimental Example 1 of the present invention is the strain reported in the article "Direct Yeast Surface Codisplay of Sequential Enzymes with Complementary Anchor Motifs: Enabling Enhanced Glycosylation of Natural Products". Specifically, it is a strain with GS115 as the chassis strain, Agα as the anchor protein, and the glycosyltransferase UGT73F24m as the reporter protein for surface display. Those skilled in the art can obtain the starting strain UGT-Agα of the present invention by combining the strain construction method described above with the conventional technical means in the fields of molecular biology and microbiology; the starting strain UGT-Agα is currently preserved in the applicant's laboratory, and the applicant undertakes to make it available to the public within 20 years from the filing date of the present invention for verifying the technical effects of the present invention.

[0042] Group 1 Embodiments, Method for Improving Cell Surface Display Efficiency of the Present Invention

[0043] This group of embodiments provides a method for improving cell surface display efficiency. All embodiments in this group have the following common features: The method for improving cell surface display efficiency includes the following steps: S1. Increase the size of the cell.

[0044] In a specific embodiment, genes in the cell are knocked out to increase the size of the cell; the genes are selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223.

[0045] In a more specific embodiment, knockout of PAS_chr2-1_0508, or PAS_chr3_0819, or PAS_chr2-2_0036, or PAS_chr2-1_0366, or PAS_chr1-4_0244, or PAS_chr3_0688, or PAS_chr1-1_0383, or PAS_chr1-4_0081, or PAS_chr4_0223 in the cell, or simultaneous knockout of the two genes PAS_chr2-1_0508 and PAS_chr2-1_0366, or the two genes PAS_chr2-1_0508 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0508 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0508 and PAS_chr4_0223, the two genes PAS_chr2-1_0366 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0366 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0366 and PAS_chr4_0223, the two genes PAS_chr1-1_0383 and PAS_chr1-4_0081, the two genes PAS_chr1-1_0383 and PAS_chr4_0223, the two genes PAS_chr1-4_0081 and PAS_chr4_0223 in the cell.

[0046] In specific embodiments, all gene names herein, including but not limited to "PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223", are unique gene symbols and locus tags that can be searched on NCBI, and those skilled in the art can find all information of the gene on NCBI.

[0047] In some specific embodiments, the knockout is performed by homologous recombination. The method of homologous recombination has the conventional technical meaning well-known to those skilled in the art. Specifically, it can be the method of homologous recombination described in the article "Direct selection of Pichia pastoris expression strains using new G418 resistance vectors".

[0048] In some embodiments, the cell is yeast;

[0049] Preferably, the yeast is Pichia pastoris;

[0050] Preferably, the Pichia pastoris is strain UGT-Agα.

[0051] Group 2 of embodiments, products of the present invention for improving cell surface display efficiency

[0052] This group of embodiments provides a product for improving cell surface display efficiency. All embodiments in this group have the following common feature: The product for improving cell surface display efficiency includes: cells with increased size.

[0053] In a specific embodiment, the cells with increased size are cells in which a gene has been knocked out; the gene is selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223.

[0054] In some embodiments, the cells with increased size are selected from: cells in which the gene PAS_chr2-1_0508, or the gene PAS_chr3_0819, or the gene PAS_chr2-2_0036, or the gene PAS_chr2-1_0366, or the gene PAS_chr1-4_0244, or the gene PAS_chr3_0688, or the gene PAS_chr1-1_0383, or the gene PAS_chr1-4_0081, or the gene PAS_chr4_0223 has been knocked out, or cells in which the two genes PAS_chr2-1_0508 and PAS_chr2-1_0366, or the two genes PAS_chr2-1_0508 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0508 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0508 and PAS_chr4_0223, the two genes PAS_chr2-1_0366 and PAS_chr1-1_0383, the two genes PAS_chr2-1_0366 and PAS_chr1-4_0081, the two genes PAS_chr2-1_0366 and PAS_chr4_0223, the two genes PAS_chr1-1_0383 and PAS_chr1-4_0081, the two genes PAS_chr1-1_0383 and PAS_chr4_0223, the two genes PAS_chr1-4_0081 and PAS_chr4_0223 have been knocked out simultaneously.

[0055] In other embodiments, the cells are yeast;

[0056] Preferably, the yeast is Pichia pastoris;

[0057] Preferably, the Pichia pastoris is the UGT-Agα strain.

[0058] In some more specific embodiments, the product for improving cell surface display efficiency is selected from: KO-1 strain with gene PAS_chr2-1_0508 knocked out, or KO-3 strain with gene PAS_chr3_0819 knocked out, or KO-4 strain with gene PAS_chr2-2_0036 knocked out, or KO-5 strain with gene PAS_chr2-1_0366 knocked out, or KO-6 strain with gene PAS_chr1-4_0244 knocked out, or KO-7 strain with gene PAS_chr3_0688 knocked out, or KO-8 strain with gene PAS_chr1-1_0383 knocked out, or KO-9 strain with gene PAS_chr1-4_0081 knocked out, or KO-10 strain of cells with gene PAS_chr4_0223 knocked out, or KO-1+KO-5 strain with both genes PAS_chr2-1_0508 and PAS_chr2-1_0366 knocked out, or KO-1+KO-8 strain with both genes PAS_chr2-1_0508 and PAS_chr1-1_0383 knocked out, KO-1+KO-9 strain with both genes PAS_chr2-1_0508 and PAS_chr1-4_0081 knocked out, KO-1+KO-10 strain with both genes PAS_chr2-1_0508 and PAS_chr4_0223 knocked out, KO-5+KO-8 strain with both genes PAS_chr2-1_0366 and PAS_chr1-1_0383 knocked out, KO-5+KO-9 strain with both genes PAS_chr2-1_0366 and PAS_chr1-4_0081 knocked out, KO-5+KO-10 strain with both genes PAS_chr2-1_0366 and PAS_chr4_0223 knocked out, KO-8+KO-9 strain with both genes PAS_chr1-1_0383 and PAS_chr1-4_0081 knocked out, KO-8+KO-10 strain with both genes PAS_chr1-1_0383 and PAS_chr4_0223 knocked out, KO-9+KO-10 strain with both genes PAS_chr1-4_0081 and PAS_chr4_0223 knocked out.

[0059] Experimental Example 1. Cell Size Adjustment

[0060] I. Construction of Single-Gene Knockout Strains

[0061] In this experimental example, the UGT-Agα strain was used as the starting strain, and the following genes: PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223 were knocked out one by one using the homologous recombination method described in the article "Direct selection of Pichia pastoris expression strains using new G418 resistance vectors". Then, through plate screening containing G418 antibiotic and yeast colony PCR verification, 10 single-gene knockout strains were finally successfully constructed. The PCR verification results are as Figure 1 shown.

[0062] II. Size Characterization of Single-Gene Knockout Strains

[0063] After all single-gene knockout strains were constructed, in this experimental example, according to the method of measuring cell size using a flow cytometer described in the article "Apparent diameter and cell density of yeast strains with different ploidy", the sizes of approximately 20,000 cells of the 10 single-gene knockout strains were measured. It was verified that there were significant differences in cell size between these 10 single-gene knockout strains and the starting strain UGT-Agα strain (P value < 0.05). These 10 single-gene knockout strains were respectively named: PAS_chr2-1_0508 (KO-1), PAS_chr1-4_0585 (KO-2), PAS_chr3_0819 (KO-3), PAS_chr2-2_0036 (KO-4), PAS_chr2-1_0366 (KO-5), PAS_chr1-4_0244 (KO-6), PAS_chr3_0688 (KO-7), PAS_chr1-1_0383 (KO-8), PAS_chr1-4_0081 (KO-9), PAS_chr4_0223 (KO-10).

[0064] The analysis of the significant differences between these 10 strains and the wild-type strain UGT-Agα is as Figure 2As shown in the figure, it can be concluded from the figure that except that the average diameter of the single-gene knockout strain KO-2 is significantly smaller than that of UGT-Agα, the average diameters of other single-gene knockout strains are significantly larger than that of UGT-Agα. In addition, compared with UGT-Agα, the single-gene knockout strains KO-5, KO-8, KO-9, and KO-10 have a greater significant difference, reaching Pvalue < 0.001. This indicates that the genes PAS_chr2-1_0366, PAS_chr1-1_0383, PAS_chr1-4_0081, and PAS_chr4_0223 have a greater impact on cell size. Among them, the cell diameters of strains KO-8 and KO-9 reach more than 3.4 μm, which is 34% larger than the cell diameter of the wild type. (Note: *P value < 0.05, **Pvalue < 0.01, ***P value < 0.001).

[0065] In addition, assuming that yeast cells are spherical, this experimental example analyzed the mean cell surface area (MSA) and mean cell volume (MCV) of these 10 strains, as Figure 3 shown. It can be concluded from the figure that compared with UGT-Agα, the MSA and MCV of the single-gene knockout strains KO-5, KO-8, KO-9, and KO-10 increase significantly. In particular, the mean cell surface areas of strains KO-8 and KO-9 are 81% and 83% larger than that of the wild-type cells, respectively, and the mean cell volumes are 145% and 148% larger, respectively.

[0066] In summary, this experimental example characterized the cell sizes of 117 single-gene knockout strains, screened and verified 10 genes with a greater impact on cell size from 117 candidate genes, namely: PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, PAS_chr4_0223. Among the 10 strains with significant changes in cell size, except for strain KO-2 which is significantly smaller, the other strains are significantly larger. In particular, strains KO-1, KO-5, KO-8, KO-9, and KO-10 have the largest average cell diameters, which are: 2.92 μm, 3.06 μm, 3.40 μm, 3.43 μm, and 3.12 μm, respectively.

[0067] II. Regulation of the cell size of Pichia pastoris

[0068] Previously, 10 genes with a greater impact on the cell size of Pichia pastoris were obtained in this experimental example. Next, this experimental example hopes to further regulate the cell size by combinatorial knockout of these genes.

[0069] III. Construction of double-gene knockout strains

[0070] According to the previous conclusion, this experimental example selected 5 strains with relatively large changes in cell size: KO-1, KO-5, KO-8, KO-9, KO-10 for the following experiments. The combinatorial strategy for double-gene knockout is as shown.

[0071] Table 1. Combinatorial strategy for double-gene knockout

[0072]

[0073] This experimental example constructed 10 double-gene knockout strains using CRISPR-Cas9 and homologous recombination method with the hygromycin phosphotransferase (hph) gene as the selection marker. The PCR electrophoresis results of the 10 double-gene knockout strains are as Figure 4 shown. IV. Size characterization of double-gene knockout strains

[0074] This experimental example also used the method of measuring cell size using a flow cytometer described in the article "Apparent diameter and cell density of yeast strains with different ploidy" to measure the sizes of approximately 20,000 yeast cells of the double-gene knockout strains, and took their average diameter, as Figure 5 shown. This experimental example can draw the following conclusions: ① Not all the average diameters of double-gene knockout strains are larger than those of single-gene knockout strains. Even compared with single-gene knockout strains, the average diameter of double-gene knockout strains is slightly decreased, such as KO-1+KO-5. ② In addition, there is no significant difference between strains KO-5+KO-9, KO-5+KO-10, KO-8+KO-9 compared with their respective single-gene knockout strains; ③ Strains KO-1+KO-8, KO-1+KO-9, KO-1+KO-10, KO-5+KO-8, KO-8+KO-10, KO-9+KO-10 show a significant increasing trend compared with one of their respective single-gene knockout strains, but there is no significant change compared with the other single-gene knockout strain. Analyzing the reasons, on the one hand, there may be certain errors in the measurement itself, resulting in the software being unable to calculate the tiny differences; on the other hand, the knockout of two genes may affect the growth or various metabolisms of the strain itself, so the expected additive effect of double-gene knockout cannot be obtained.

[0075] In this experimental example, the significance analysis of these 10 double-gene knockout strains and UGT-Agα was first carried out as follows Figure 6 As shown. It can be concluded from the figure that except that the average diameter of the double-gene knockout strain KO-1+KO-5 has no significant difference compared with UGT-Agα, the other 9 double-gene knockout strains all have significant differences from UGT-Agα (P value < 0.05), and their average diameters are all significantly larger than that of UGT-Agα. Among them, the double-gene knockout strain KO-9+KO-10 has the largest average diameter of 3.63 μm, which is the largest cell diameter obtained after the regulation of this experimental example

[0076] In addition, similar to the analysis of single-gene knockout strains in this experimental example, the MSA and MCV of these 10 double-gene knockout strains were calculated, as shown Figure 7 As shown. It can be concluded from the figure that except that there is no significant difference between the double-gene knockout strains KO-1+KO-5 and KO-5+KO-10 and UGT-Agα, the MSA and MCV of other strains increase significantly. Among them, the average cell surface area and average cell volume of KO-9+KO-10 are increased by 1.05 and 1.94 times respectively compared with the wild type

[0077] By using the methods of single-gene knockout and gene combination knockout, this experimental example finally obtained 19 strains with significant changes in the average cell diameter. Except that the strain KO-2 decreased significantly, the other strains increased significantly. Among them, the single-gene knockout strains KO-1, KO-5, KO-8, KO-9, KO-10 and the double-gene knockout strain KO-9+KO-10 obtained the largest average cell diameter, which are 2.92 μm, 3.06 μm, 3.40 μm, 3.43 μm, 3.12 μm and 3.63 μm respectively. It shows that the knockout of these genes can regulate the cell size to a certain extent. Then, in order to further illustrate that the change of cell size is not caused by the environment, this experimental example measured the cell dry weight of the 6 strains with the largest change in average diameter mainly according to the measurement method of cell dry weight recorded in the article "Metabolic Engineering Promotes the Synthesis of Carotenoids in Pichia pastoris", as shown Figure 8 As shown. It can be seen from the figure that the dry weight of a single cell increases from 2.62×10 -6 μg to 3.53×10 -6 μg, 3.68×10 -6 μg, 4.45×10 -6 μg, 4.37×10 -6 μg, 3.81×10 -6 μg, 4.77×10 -6μg, indicating that the changes in the cell size of these strains are indeed regulated by the corresponding genes, rather than caused by absorbing moisture in the environment.

[0078] After obtaining the strains with changed cell sizes by knocking out the relevant genes in this experimental example, this experimental example wanted to explore whether the knockout of these genes would affect the cell growth state. Therefore, according to the measurement method of cell growth curve recorded in the article "Preliminary Exploration on Strengthening β-Carotene Synthesis by Regulating the Metabolic Pathway of Saccharomyces cerevisiae", the cell growth curves of 10 double-gene knockout strains were measured, as Figure 9 shown. It can be seen from the figure that the OD of these 10 strains within 72 h 600 has no significant difference, indicating that the knockout of these genes will not cause growth defects in the cells.

[0079] To sum up, in the third part of this experimental example, the cell size of Pichia pastoris was further regulated by the method of double-gene knockout, and finally 9 strains with significantly larger cell diameters were obtained, namely: KO-1+KO-8, KO-1+KO-9, KO-1+KO-10, KO-5+KO-8, KO-5+KO-9, KO-5+KO-10, KO-8+KO-9, KO-9+KO-10. And the cell dry weights of the 5 strains with the largest average cell diameters in the single-gene knockout strains and double-gene knockout strains were measured, proving that the increase in the cell size of these strains is indeed caused by the regulation of the relevant genes, rather than being affected by the environment. In addition, this experimental example measured the growth curves of 10 double-gene knockout strains within 72 h, and the results showed that the knockout of these genes will not affect the normal growth of the cells.

[0080] Experimental Example 2: Verification of improved cell surface display efficiency

[0081] I. Application of Pichia pastoris size regulation in yeast surface display

[0082] As mentioned above, in this experimental example, the glycosyltransferase UGT73F24m was used as the reported protein for display to explore the effect of Pichia pastoris size regulation on its display efficiency. UGT73F24m is a mutant constructed by the research group in the early stage. This enzyme can catalyze the conversion of glycyrrhetinic acid (GA) into 3-O-glucosyl-glycyrrhetinic acid (GLMG), as shown in Figure 10.

[0083] II. Characterization of the display amount and activity of single-gene knockout strains

[0084] First, in this experimental example, 10 single-gene knockout strains obtained in Experimental Example 1 were used as the research objects, and the amount of displayed enzyme of the single-gene knockout strains was measured using enzyme-linked immunosorbent assay (ELISA). As Figure 11 shown, the results indicate that except for the amount of enzyme displayed per single cell of strain KO-2, which has no significant difference from UGT-Agα, the displayed amounts of the remaining strains all increased significantly. Among them, the displayed amounts of KO-1, KO-5, KO-8, KO-9, and KO-10 are relatively high, and the number of enzyme molecules displayed per single cell is 2.36×10 4 , 2.50×10 4 , 2.51×10 4 , 2.77×10 4 , 2.34×10 4 (pieces) respectively. Compared with the starting strain UGT-Agα (1.79×10 4 pieces), the displayed amounts increased by 31.69±4.61%, 39.43±5.67%, 39.90±8.85%, 54.70±0.17%, and 30.73±6.21% respectively. It can be concluded that when the cell size increases, the number of enzyme molecules displayed on the surface of a single yeast cell also increases, indicating that the strategy of regulating cell size to a certain extent can indeed effectively regulate the amount of enzyme displayed on the surface.

[0085] Then, in this experimental example, the catalytic activity of the surface-displayed enzyme was explored, as Figure 12 shown. The results indicate that except for the concentration of GLMG generated by per single cell of strain KO-2, which has no significant difference compared with UGT-Agα, the concentration of GLMG generated by per single cell of other single-gene knockout strains all increased significantly. Among them, the concentrations of GLMG generated by the enzymes displayed on per single cell of KO-1, KO-5, KO-8, KO-9, and KO-10 are relatively high, which are 13.91, 14.01, 15.72, 15.85, and 13.79 (×10 -12 μM) respectively. Compared with the concentration of GLMG generated by the enzyme displayed on per single cell of the starting strain UGT-Agα (8.14×10 -12 μM), the concentrations increased by 70.84±1.49%, 72.06±6.92%, 93.12±7.35%, 94.70±3.64%, and 69.36±3.22% respectively. It can be concluded that when the amount of enzyme displayed on per single cell increases, the activity of the enzyme displayed on the surface of a single cell also increases. The change trend of the displayed enzyme activity is consistent with the displayed amount, indicating that increasing the displayed amount is an effective strategy to improve the enzyme activity and display efficiency. And the strategy of regulating cell size proposed in this experimental example can indeed effectively regulate the amount of enzyme displayed on the cell surface, thereby affecting the enzyme activity and display efficiency.

[0086] In summary, in the second part of this experimental example, the effects of 10 single-gene knockout strains with significantly changed cell sizes, namely KO-1 to KO-10, on the display amount and activity of UGT73F24m displayed on the yeast surface were specifically explored. Among them, the amount of enzyme displayed per single cell and the catalytic activity of the enzyme displayed per single cell of strains KO-1, KO-5, KO-8, KO-9, and KO-10 were relatively high. In addition, from Figure 13 it can be seen that there is no simple monotonic linear relationship between cell size and the amount of enzyme displayed per single cell. That is, compared with other strains, even if the average cell diameter of a certain strain is larger, it does not mean that the amount of enzyme displayed by this single cell is larger. The reasons for this situation may be affected by many aspects such as detection errors. Therefore, the relationship between cell size and the number of enzyme molecules displayed may need to be further studied in depth.

[0087] III. Display amount and activity characterization of double-gene knockout strains

[0088] Next, in this experimental example, the amount of enzyme displayed per single cell of double-gene knockout strains was also measured using the ELISA method. As Figure 14 shown, the results indicate that the amounts of enzyme displayed per single cell of these 10 strains were 79.82%, 164.02%, 132.49%, 165.47%, 159.41%, 165.15%, 135.92%, 165.40%, 144.04%, and 174.54% of the starting strain UGT-A, respectively.

[0089] Immediately afterwards, the catalytic activity of the enzyme was explored in this experiment, as Figure 15 shown. The results indicate that except that the concentration of GLMG produced per single cell of strain KO-1+KO-5 was significantly lower than that of UGT-Agα, the concentrations of GLMG produced per single cell of the remaining 9 strains increased significantly, which were 16.56, 14.33, 16.82, 16.23, 16.85, 12.88, 17.18, 14.89, and 17.81 (μM / cell), respectively. Compared with the starting strain UGT-A, the concentrations of GLMG produced per single cell increased by 103.39±8.72%, 76.09±0.93%, 99.36±3.66%, 106.96±4.29%, 58.27±3.13%, 111.05±3.53%, 82.96±4.53%, 118.80±2.16%, respectively.

[0090] In the third part of this experimental example, the application of 10 strains with further regulated cell sizes in yeast surface display was specifically explored. From the results, except for the strain KO-1+KO-5, the amount of enzyme displayed per single cell and the enzyme catalytic activity per single cell of other strains were improved to varying degrees. Immediately afterwards, in this experimental example, the strain KO-9+KO-10 with the largest cell size, the highest amount of enzyme displayed per single cell, and the highest enzyme activity was compared with its corresponding single gene knockout strain to further explore the effect of cell size change on the display efficiency, as Figure 16 shown. It can be concluded from this that compared with its corresponding single gene knockout strain, the double gene knockout strain KO-9+KO-10 has a larger cell size, a higher display amount, and a higher display enzyme activity, further indicating that the increase in cell size can improve the display efficiency on the yeast surface to a certain extent.

[0091] Although the present invention has been described in detail with general descriptions and specific implementation manners in the foregoing text, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for improving cell surface display efficiency, characterized in that: The method comprises the following steps: S1. increasing the size of the cells.

2. A method for improving cell surface display efficiency according to claim 1, characterized in that: Knocking out a gene in the cell to increase the size of the cell; the gene is selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, and PAS_chr4_0223.

3. A method for improving cell surface display efficiency according to claim 2, characterized in that: Knock out PAS_chr2-1_0508, or PAS_chr3_0819, or PAS_chr2-2_0036, or PAS_chr2-1_0366, or PAS_chr1-4_0244, or PAS_chr3_0688, or PAS_chr1-1_0383, or PAS_chr1-4_0081, or PAS_chr4_0223 in the cell, or knock out the double genes PAS_chr2-1_0508 and PAS_chr2-1_0366, or the double genes PAS_chr2-1_0508 and PAS_chr1-1_0383, or the double genes PAS_chr2-1_0508 in the cell at the same time and PAS_chr1-4_0081, double genes PAS_chr2-1_0508 and PAS_chr4_0223, double genes PAS_chr2-1_0366 and PAS_chr1-1_0383, double genes PAS_chr2-1_0366 and PAS_chr1-4_0081, double genes PAS_chr2-1_0366 and PAS_chr4_0223, double genes PAS_chr1-1_0383 and PAS_chr1-4_0081, double genes PAS_chr1-1_0383 and PAS_chr4_0223, double genes PAS_chr1-4_0081 and PAS_chr4_0223.

4. A method for improving cell surface display efficiency according to claim 2 or 3, characterized in that: The knockout is carried out by homologous recombination.

5. A method for improving cell surface display efficiency according to any one of claims 1 to 4, characterized in that: The cell is yeast; and / or, the yeast is Pichia pastoris; And / or, the Pichia pastoris is a UGT-Agα strain.

6. A product for improving the efficiency of cell surface display, characterized in that: include: Cells that increase in size.

7. A product for improving cell surface display efficiency according to claim 6, characterized in that: The cells with increased size are cells with knocked-out genes; the genes are selected from the group consisting of PAS_chr2-1_0508, PAS_chr1-4_0585, PAS_chr3_0819, PAS_chr2-2_0036, PAS_chr2-1_0366, PAS_chr1-4_0244, PAS_chr3_0688, PAS_chr1-1_0383, PAS_chr1-4_0081, and PAS_chr4_0223.

8. A product for improving cell surface display efficiency according to claim 7, characterized in that: The cells with increased size are selected from: cells with knocked-out gene PAS_chr2-1_0508, or gene PAS_chr3_0819, or gene PAS_chr2-2_0036, or gene PAS_chr2-1_0366, or gene PAS_chr1-4_0244, or gene PAS_chr3_0688, or gene PAS_chr1-1_0383, or gene PAS_chr1-4_0081, or gene PAS_chr4_0223, or cells with knocked-out double genes PAS_chr2-1_0508 and PAS_chr2-1_0366, or double genes PAS_chr2-1_0508 and PAS_chr1-1_0383, or double genes PAS_chr4_0223. chr2-1_0508 and PAS_chr1-4_0081, double genes PAS_chr2-1_0508 and PAS_chr4_0223, double genes PAS_chr2-1_0366 and PAS_chr1-1_0383, double genes PAS_chr2-1_0366 and PAS_chr1-4_0081, double genes PAS_chr2-1_0366 and PAS_chr4_0223, double genes PAS_chr1-1_0383 and PAS_chr1-4_0081, double genes PAS_chr1-1_0383 and PAS_chr4_0223, double genes PAS_chr1-4_0081 and PAS_chr4_0223.

9. A product for improving cell surface display efficiency according to claim 8, characterized in that: The cell is yeast; and / or, the yeast is Pichia pastoris; And / or, the Pichia pastoris is a UGT-Agα strain.

10. A product for improving cell surface display efficiency according to any one of claims 6 to 9, characterized in that: Selected from: KO-1 strain with PAS_chr2-1_0508 knocked out, KO-3 strain with PAS_chr3_0819 knocked out, KO-4 strain with PAS_chr2-2_0036 knocked out, KO-5 strain with PAS_chr2-1_0366 knocked out, KO-6 strain with PAS_chr1-4_0244 knocked out, KO-7 strain with PAS_chr3_0688 knocked out, or KO-8 strain with PAS_chr1-1_0383 knocked out. KO-8 strain, or KO-9 strain in which the gene PAS_chr1-4_0081 was knocked out, or KO-10 strain in which the gene PAS_chr4_0223 was knocked out, or KO-1+KO-5 strain in which the double genes PAS_chr2-1_0508 and PAS_chr2-1_0366 were knocked out at the same time, or KO-1+KO-8 strain in which the double genes PAS_chr2-1_0508 and PAS_chr1-1_0383 were knocked out at the same time, or KO-1+KO-8 strain in which the double genes PAS_chr2-1_050 8 and PAS_chr1-4_0081, KO-1+KO-10 strains in which the double genes PAS_chr2-1_0508 and PAS_chr4_0223 were knocked out at the same time, KO-5+KO-8 strains in which the double genes PAS_chr2-1_0366 and PAS_chr1-1_0383 were knocked out at the same time, KO-5+KO-9 strains in which the double genes PAS_chr2-1_0366 and PAS_chr1-4_0081 were knocked out at the same time, and KO-6+KO-7 strains in which the double genes PAS_chr2-1_0508 and PAS_chr4_0223 were knocked out at the same time. The KO-5+KO-10 strain with S_chr2-1_0366 and PAS_chr4_0223, the KO-8+KO-9 strain with the double genes PAS_chr1-1_0383 and PAS_chr1-4_0081 knocked out at the same time, the KO-8+KO-10 strain with the double genes PAS_chr1-1_0383 and PAS_chr4_0223 knocked out at the same time, and the KO-9+KO-10 strain with the double genes PAS_chr1-4_0081 and PAS_chr4_0223 knocked out at the same time.

Images