Method for improving expression quantity of AW-YGL lysozyme in GS115 pichia pastoris based on CRISPRCas9 technology

Abstract

The invention discloses a method for improving the expression quantity of AW-YGL lysozyme in GS115 pichia pastoris based on a CRISPRCas9 technology, and relates to the technical field of bioengineering, and the method is technically characterized in that related genes XP and YPS of pichia pastoris cell walls alpha are knocked out by utilizing a CRISPR / cas9 technology, so that the expression quantity of the AW-YGL lysozyme in the GS115 pichia pastoris is improved, the extraction process of the lysozyme is simplified, and the yield of the lysozyme is improved. The antibacterial effect is enhanced, so that the application range in the medicine industry, the animal husbandry industry and the food industry is expanded.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a method for increasing the expression level of AW-YGL lysozyme in GS115 Pichia pastoris based on CRISPR / Cas9 technology. Background Art

[0002] The widespread use of antibiotics has led to the emergence of super-resistant bacteria, a serious threat to human health and sustainable economic development. China completely banned the addition of growth-promoting antibiotics to feed in 2020 to reduce the emergence of drug-resistant bacteria and the presence of antibiotic residues in livestock and poultry products. Therefore, the search for alternatives to feed antibiotics has become a current research hotspot.

[0003] Lysozyme, also known as muramidase or N-acetylmuramidase, is a naturally occurring antibacterial enzyme. Lysozyme acts on the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the peptidoglycan (PG) of bacterial cell walls, disrupting the integrity of the bacterial cell wall and causing bacterial cell rupture, thereby exerting its antibacterial properties. Lysozyme is a natural antibacterial substance widely used in the livestock, pharmaceutical, and food industries. It can be used to treat oral ulcers and pharyngitis, kill pathogenic microorganisms in livestock and poultry, alleviate inflammation, and enhance immunity and growth efficiency. In children's toothpaste, it can inhibit oral pathogens and prevent dental caries. Natural lysozyme is effective against Gram-positive bacteria but less effective against Gram-negative bacteria. Its low content and complex extraction process limit its application.

[0004] To this end, the present invention aims to provide a method for increasing the expression level of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems and provide a method for increasing the expression of AW-YGL lysozyme in GS115 Pichia pastoris based on CRISPR / Cas9 technology. The present invention uses CRISPR / Cas9 technology to knock out the Pichia pastoris cell wall α-related genes XP and YPS, thereby increasing the expression of AW-YGL lysozyme in GS115 Pichia pastoris.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology, the method comprising the following steps:

[0008] S1. Construction of recombinant plasmids: Using plasmid JME4599 as a template, synthesize fragments YPS1, YPS2, XP1, and XP2 containing sgRNA genes. Use homologous recombination to construct CRISPR / cas9 knockout plasmids JME-XP and JME-YPS to obtain recombinant plasmids. Transform the recombinant plasmids into E. coli competent cells TOP10, screen for positive transformants using LB solid medium containing 1 mg / L kanamycin, and perform colony PCR verification using primers YPS1-F, YPS1-R, XP1-F, and XP1-R. Transformants that are verified as positive are sent for sequencing. Transformants that have been sequenced correctly are inoculated into 10 mL of LB liquid medium for amplification and culture, and the recombinant plasmid is extracted using a plasmid extraction kit.

[0009] S2. Extract the genome of Pichia pastoris GS115, download 1000 bp upstream and downstream of the XP (XP_002492593) and YPS (8196641) genes from NCBI, design primers XP-Donar1-F, XP-Donar1-R, XP-Donar2-F, XP-Donar2-R, YPS-Donar1-F, YPS-Donar1-R, YPS-Donar2-F, and YPS-Donar2-R, and then synthesize 2000 bp of Donar-XP and 2000 bp of Donar-YPS by overlapping PCR;

[0010] S3. Transform GS115AW-YGL competent yeast with the recombinant plasmid and screen for positive transformants: 100ng of circular plasmid JME-XP, 200ng of Donar-XP, and 100ng of circular plasmid JME-YPS and 200ng of Donar-YPS fragment were electroporated into competent Pichia pastoris GS115-AW-YGL. Positive transformants were screened using solid YPD medium containing 1mg / L G418. The selected positive transformants were streaked twice on a G418 plate, and then streaked on a non-resistant YPD plate to remove the JME plasmid in the cells until they could no longer grow on the plate containing the G418 antibiotic. The genome of the selected positive transformants was extracted, and the positive transformants were verified by yeast colony PCR using the genome as a template and XP-Donar1-F, XP-Donar2-R, YPS-Donar1-F, and YPS-Donar2-R as primers. The positive transformants were then picked.

[0011] S4. Fermentation expression of recombinant yeast: The positive transformants picked in S3 were inoculated into 200 mL of liquid YPD medium and fermented at 30°C and 200 rpm for 120 h to obtain AW-YGL.

[0012] Compared with the existing technology, this solution has the following beneficial effects:

[0013] The present invention utilizes CRISPR / Cas9 technology to knock out the Pichia pastoris cell wall α-related genes XP and YPS, thereby increasing the expression level of AW-YGL lysozyme in Pichia pastoris GS115 and simplifying the extraction process of lysozyme, making its inhibitory effect on Gram-positive bacteria more prominent and enhancing its antibacterial effect, thereby expanding its application range in the pharmaceutical, animal husbandry, and food industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Flowchart of the method for expressing AW-YGL lysozyme in Pichia pastoris GS115 according to an embodiment of the present invention;

[0015] Figure 2 Schematic diagram of the yeast colony PCR verification results of CRISPR knockout of XP and YPS in an embodiment of the present invention, wherein M is a DNA marker; 1 is before XP knockout; 2 is before YPS knockout; 3 is after XP knockout; 4 is after YPS knockout;

[0016] Figure 3 Schematic diagram of the enzyme activity results of the fermentation broth of the knockout strain in the embodiment of the present invention;

[0017] Figure 4 Schematic diagram of the recombinant protein content in the fermentation broth of the knockout strain in the embodiment of the present invention;

[0018] Figure 5 Schematic diagram of the WB analysis results of the recombinant protein of the knockout strain in the embodiment of the present invention. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0020] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0021] Example: Research Methods

[0022] 1. Materials

[0023] 1.1. Strains and plasmids

[0024] Pichia pastoris GS115-AW-YGL was preserved in our laboratory, the amino acid sequence of AW is AWVAWK, and YGL (AMR70500.1) is yak stomach lysozyme; Escherichia coli TOP10 was purchased from Novazonics; and the expression vector JME4599 was purchased from Invitrogen.

[0025] 1.2. Experimental reagents and instruments

[0026] PCR product purification kits, agarose gel recovery kits, and plasmid extraction kits were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; homologous recombination kits were purchased from Sino-US Taihe Biotechnology Co., Ltd.; SDS-PAGE kits were purchased from Changzhou Boyi Biotechnology Co., Ltd.; BCA protein concentration assay kits were purchased from Beyotime Biotechnology Co., Ltd.; lysozyme activity assay kits were purchased from Suzhou Grace Biotechnology Co., Ltd.; kanamycin and G418 were purchased from Shanghai Maokang Biotechnology Co., Ltd.; yeast extract and peptone were purchased from Oxoid; other conventional reagents were domestically produced analytical grade.

[0027] Culture medium

[0028] LB medium: Tryptone: 10 g, Yeast Extract: 5 g, Sodium Chloride (NaCl): 10 g, Distilled Water: 1 L, Solid Medium plus Agar: 15 g. Adjust the pH to 7.0-7.2 with NaOH or HCl. Sterilize by autoclaving at 121°C for 15 minutes.

[0029] YPD medium: Yeast extract: 10g, peptone: 20g, glucose: 20g, distilled water: 1L, and agar: 15g for solid medium. Add yeast extract and peptone to distilled water and stir to dissolve. Add glucose and stir until completely dissolved. Adjust the pH to 6.5 with NaOH or HCl. Sterilize by autoclaving at 121°C for 15 minutes.

[0030] Primer sequences

[0031] Table 1 Primer sequence list

[0032]

[0033]

[0034] Table 2 sgRNA table

[0035]

[0036] 2. Experimental Methods

[0037] 2.1. Construction of recombinant plasmids

[0038] Primers YPS1-F, YPS1-R, YPS2-F, YPS2-R, XP1-F, XP1-R, XP2-F, and XP2-R were designed. Using plasmid JME4599 as a template, fragments YPS1, YPS2, XP1, and XP2 containing sgRNA genes were synthesized. CRISPR / Cas9 knockout plasmids JME-XP and JME-YPS were constructed using homologous recombination, using the same homologous recombination system as described in Table 3. The recombinant plasmids were transformed into competent E. coli TOP10 cells. Positive transformants were screened using LB solid medium supplemented with 1 mg / L kanamycin. Colony PCR was performed using primers YPS1-F, YPS1-R, XP1-F, and XP1-R, and positive transformants were sent for sequencing. Transformants that were sequenced correctly were cultured in 10 mL of LB liquid medium and the recombinant plasmids were extracted using a plasmid extraction kit.

[0039] Table 3 Homologous recombination system

[0040]

[0041] The Pichia pastoris GS115 genome was extracted, and 1000 bp upstream and downstream of the XP (XP_002492593) and YPS (8196641) genes were downloaded from NCBI. Primers XP-Donar1-F, XP-Donar1-R, XP-Donar2-F, XP-Donar2-R, YPS-Donar1-F, YPS-Donar1-R, YPS-Donar2-F, and YPS-Donar2-R were designed. 2000 bp of Donar-XP and 2000 bp of Donar-YPS were then synthesized by overlapping PCR. Table 4 shows the overlapping PCR reaction system, and Table 5 shows the overlapping PCR reaction steps.

[0042] The method for extracting the GS115 genome is as follows:

[0043] (1) Pichia pastoris GS115 was streaked onto a YPD plate and grown at 30°C for 2 days.

[0044] (2) Pick a single clone from the plate and transfer it to 5 mL of YPD medium. Incubate at 30°C and 200 rpm for 12 h.

[0045] (3) Centrifuge at 3500 g for 5 min to collect 1 mL of the bacterial solution, discard the supernatant, add 30 μL of STES buffer, and mix thoroughly using an oscillating mixer;

[0046] (4) Add 200 μL ddH2O and 200 μL phenol-chloroform and mix thoroughly using a shaker;

[0047] (5) Centrifuge at 12000g for 5 min and collect 100 μL of supernatant;

[0048] (6) Add 250 μL of pre-cooled anhydrous ethanol and 10 μL of 3 mol / L, pH 5.2 sodium acetate and mix well;

[0049] (7) Centrifuge at 12000g for 3 min and discard the supernatant;

[0050] (8) Add 500 μL of pre-cooled 70% ethanol and mix thoroughly;

[0051] (9) Centrifuge at 12000g for 2 min, discard the supernatant, and air dry;

[0052] (10) Add 50 μL of ddH2O and mix for subsequent use.

[0053] Table 4 Overlapping PCR reaction system

[0054]

[0055] Table 5 Overlap PCR reaction steps

[0056]

[0057] 2.2. Transformation of recombinant plasmid into GS115AW-YGL competent yeast and screening of positive transformants

[0058] 100 ng of the circular plasmid JME-XP, 200 ng of Donar-XP, 100 ng of the circular plasmid JME-YPS, and 200 ng of the Donar-YPS fragment were electroporated into competent Pichia pastoris GS115-AW-YGL. Positive transformants were screened using solid YPD medium supplemented with 1 mg / L G418. Transformants were streaked twice onto G418 plates and then onto non-resistant YPD plates to remove the JME plasmid from the cells until they failed to grow on plates supplemented with G418. The genome of the selected transformants was extracted and verified by yeast colony PCR using the genome as a template and primers XP-Donar1-F, XP-Donar2-R, YPS-Donar1-F, and YPS-Donar2-R. Positive transformants were selected.

[0059] 2.3. Fermentation expression of recombinant yeast

[0060] The selected positive transformants were inoculated into 200 mL of liquid YPD medium and fermented at 30°C and 200 rpm for 120 h to produce AW-YGL.

[0061] 2.4. Qualitative and quantitative analysis of expressed proteins

[0062] Take 1 mL of the supernatant solution and centrifuge at 12,000 rpm for 2 minutes. Aspirate the supernatant for SDS-PAGE gel electrophoresis analysis. Mix 10 μL of the supernatant with 50 μL of loading buffer and heat at 98°C for 5 minutes to prepare the electrophoresis sample. Prepare a 15% separating gel and a 5% stacking gel. Concentrate at 120V for 30 minutes, then separate at 180V for 90 minutes. Stain with Coomassie Brilliant Blue overnight, destain with acetic acid, and observe the protein electrophoresis results.

[0063] Table 6 SDS-PAGE gel formula

[0064]

[0065] After SDS-PAGE protein electrophoresis, remove the gel sheet, remove the stacking gel, and soak a suitable size filter paper in transfer buffer. Then, activate the PVDF membrane in methanol. Place six filter papers, the PVDF membrane, the SDS-PAGE gel, and six filter papers in the transfer apparatus in that order. Remove any bubbles and transfer the proteins from the SDS-PAGE gel to the PVDF membrane at 25V for 30 minutes. After transfer, block the PVDF membrane in 5% skim milk for 3 hours. Wash it three times with TBST (10 minutes each). Incubate the PVDF membrane with the primary antibody incubation solution for 1 hour. After incubation, wash it three times with TBST (10 minutes each). Incubate the PVDF membrane with the secondary antibody incubation solution for 1 hour. Wash it again with TBST (10 minutes each). Develop the membrane with Western ECL substrate and photograph it.

[0066] 2.5. Lysozyme Activity Assay and Protein Purification

[0067] Lysozyme activity in the fermentation supernatant was measured using a lysozyme activity assay kit. Lysozyme can degrade turbid bacterial cultures of a certain concentration, reducing turbidity and increasing transmittance. Lysozyme activity can be measured by changes in absorbance. For detailed procedures, refer to the Lysozyme Assay Kit instructions from Suzhou Green Biotechnology Co., Ltd.

[0068] The fermentation supernatant was purified using a histidine-tagged protein purification method. A nickel column was selected as the filling material. The impurities in the fermentation supernatant were first eluted with low-concentration imidazole buffer solutions of 20 mM, 50 mM, 100 mM, and 200 mM. The recombinant protein was then eluted and recovered with a 500 mM imidazole buffer solution. Table 7 shows the composition of the imidazole buffer solution.

[0069] Table 7 Imidazole buffer solution composition

[0070]

[0071] Protein quantification: The Bicinchoninic Acid (BCA) method was used to determine the protein content of the purified fermentation supernatant. A gradient of bovine serum albumin (BSA) standard solutions was prepared, and the A562 was measured. A protein standard curve was then drawn, and the content of the purified protein in the fermentation broth was calculated based on the curve. For detailed procedures, please refer to the BCA Protein Concentration Assay Kit instruction manual from Beyotime Biotechnology.

[0072] 3. Research Results

[0073] 3.1. Construction and verification of recombinant expression vector

[0074] Knockout plasmids JME-XP and JME-YPS for the cell wall glucan synthesis-related genes XP and YPS were constructed and electrotransformed together with 1000 bp DonarDNA-XP and DonarDNA-YPS into competent Pichia pastoris GS115-AW-YGL. Positive transformants were screened using YPD medium containing 1 mg / L G418, and colony PCR verification was performed using XP1-F, XP2-R, YPS1-F, and YPS2-R. Figure 2 Yeast colony PCR verification before and after CRISPR knockout of XP and YPS, Figure 2 Middle 1 is the amplified fragment before XP knockout using XP1-F and XP2-R as upstream and downstream primers, and its band position is close to the corresponding position of the 3980 bp gene; Figure 2 Middle 3 is the amplified fragment after XP was knocked out using XP1-F and XP2-R as upstream and downstream primers. The position of the band is close to the corresponding position of the 2000 bp gene; Figure 2 Middle 2 is the amplified fragment using YPS1-F and YPS2-R as upstream and downstream primers before YPS knockout, and its band position is close to the corresponding position of the 3895 bp gene; Figure 2 Figure 4 is the amplified fragment after YPS knockout using YPS1-F and YPS2-R as upstream and downstream primers. The band position is close to the corresponding position of the 2000 bp gene; therefore, it can be determined that the XP and YPS genes were successfully knocked out.

[0075] 3.2. Enzyme activity and protein analysis of fermentation supernatant

[0076] The yeast fermentation broth was centrifuged and the supernatant was collected. The lysozyme activity was determined using a lysozyme activity assay kit. Figure 3 The AW-YGL lysozyme activity in the fermentation supernatant of the strains with CRISPR knockout of target genes such as XP and YPS was 613333U / L and 688000U / L, respectively. The activity in the fermentation supernatant of the control group GS115 was 301818U / L. The recombinant lysozyme in the fermentation supernatant was purified using the histidine tag protein purification method, and the concentration of the purified protein was determined using the BCA protein concentration assay kit. Figure 4 The recombinant lysozyme content in the fermentation supernatant of the strains with target genes such as XP and YPS knocked out by CRISPR was 0.31 mg / L and 0.39 mg / L, respectively. The recombinant lysozyme content in the fermentation supernatant of the control group GS115 was 0.27 mg / L. The WB protein quantitative analysis was performed. Figure 5 Western blot analysis of recombinant lysozyme in the fermentation supernatant of strains with CRISPR knockout of target genes such as XP and YPS. The recombinant protein blot of the knockout gene was darker than that of the control group, and the recombinant protein blot of the YPS gene knockout was the darkest.

[0077] 4. Discussion

[0078] In this study, CRISPR / Cas9 gene editing technology was used to knock out the cell wall synthesis-related genes XP and YP in the yeast GS115 to improve the recombinant expression efficiency of AW-YGL. Results showed that knocking out both XP and YPS increased the secretion efficiency of AW-YGL. Knocking out the XP gene increased the secretion efficiency of AW-YGL to 613,333 U / L, while knocking out the YPS gene increased the secretion efficiency to 688,000 U / L, successfully achieving high-efficiency expression of lysozyme in Pichia pastoris.

[0079] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR-Cas9 technology, characterized by: The method comprises the following steps: S1. Construction of recombinant plasmids: Using plasmid JME4599 as a template, synthesize fragments YPS1, YPS2, XP1, and XP2 containing sgRNA genes, and construct CRISPR / cas9 knockout plasmids JME-XP and JME-YPS by homologous recombination to obtain recombinant plasmids; The recombinant plasmid was transformed into competent E. coli TOP10 cells. Positive transformants were screened using LB solid medium containing 1 mg / L kanamycin. Colony PCR was performed using primers YPS1-F, YPS1-R, XP1-F, and XP1-R, and positive transformants were sent for sequencing. Transformants that were sequenced correctly were inoculated into 10 mL of LB liquid medium for amplification and culture. The recombinant plasmid was extracted using a plasmid extraction kit. S2. The genome of Pichia pastoris GS115 was extracted. Based on the 1000 bp upstream and downstream of the XP and YPS genes, primers XP-Donar1-F, XP-Donar1-R, XP-Donar2-F, XP-Donar2-R, YPS-Donar1-F, YPS-Donar1-R, YPS-Donar2-F, and YPS-Donar2-R were designed. Then, 2000 bp of Donar-XP and 2000 bp of Donar-YPS were synthesized by overlapping PCR. S3. Transform GS115AW-YGL competent yeast with the recombinant plasmid and screen for positive transformants: 100 ng of the circular plasmid JME-XP, 200 ng of Donar-XP, and 100 ng of the circular plasmid JME-YPS and 200 ng of the Donar-YPS fragment were electroporated into competent Pichia pastoris GS115-AW-YGL, respectively; positive transformants were screened using solid YPD medium containing 1 mg / L G418, and the genomes of the screened positive transformants were extracted. Positive transformants were verified by yeast colony PCR using the genomes as templates and XP-Donar1-F, XP-Donar2-R, YPS-Donar1-F, and YPS-Donar2-R as primers, and positive transformants were picked; S4. Fermentation expression of recombinant yeast: The positive transformants picked in S3 were inoculated into 200 mL of liquid YPD medium and fermented at 30°C and 200 rpm for 120 h to obtain AW-YGL.

2. The method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology according to claim 1, characterized in that: In S1, the recombinant plasmid is transformed into Escherichia coli competent cells TOP10, and positive transformants are screened using LB solid medium containing 1 mg / L kanamycin. Colony PCR verification is performed using primers YPS1-F, YPS1-R, XP1-F, and XP1-R, and transformants that are verified as positive are sent for sequencing; transformants with correct sequencing are inoculated into 10 mL LB liquid medium for amplification and culture, and the recombinant plasmid is extracted using a plasmid extraction kit.

3. The method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology according to claim 1, characterized in that: The XP and YPS genes were downloaded from NCBI.

4. The method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology according to claim 1, characterized in that: The method for extracting the GS115 genome is as follows: S1. Pick Pichia pastoris GS115, streak it onto a YPD solid plate, and grow it at 30°C for 2 days; S2. Pick a single clone from the plate and transfer it to 5 mL of YPD medium. Incubate at 30°C and 200 rpm for 12 h. S3. Centrifuge at 3500g for 5 min to collect 1 mL of the bacterial solution, discard the supernatant, add 30 μL of STES buffer, and shake on a shaker to mix thoroughly. S4, add 200 μL ddH2O and 200 μL phenol chloroform, and shake on a shaker to mix; S5, centrifugation at 12000g for 5 min, and 100 μL of supernatant was collected; S6. Add 250 μL of pre-cooled anhydrous ethanol and 10 μL of 3 mol / L, pH 5.2 sodium acetate and mix well. S7, centrifugation at 12000g for 3 min, discarding the supernatant; S8. Add 500 μL of pre-chilled 70% ethanol and mix thoroughly. S9, centrifuge at 12000g for 2 min, discard the supernatant, and air dry; S10. Add 50 μL of ddH2O and mix to obtain the GS115 genome solution.

5. The method for increasing the expression of AW-YGL lysozyme in Pichia pastoris GS115 based on CRISPR Cas9 technology according to claim 1, characterized in that: In S3, positive transformants were screened using solid YPD medium containing 1 mg / L G418. The screened positive transformants were streaked twice on G418 plates and then streaked on non-resistant YPD plates to remove the JME plasmid in the cells until the cells could no longer grow on the plate containing G418 antibiotic. The genome of the screened positive transformants was then extracted.

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