A Pichia pastoris genetic engineering bacterium for highly efficient surface display of an enzyme based on an adhesion protein, a construction method thereof, and applications thereof

Abstract

The present invention discloses a Pichia pastoris genetically engineered bacterium with efficient surface display of enzymes based on adhesion proteins, a construction method thereof, and an application in immobilized continuous catalysis. Adhesion protein genes, β-galactosidase gene LacA, and cell wall anchor protein gene Pir1p are heterologously expressed in the starting strain Pichia pastoris. The Pichia pastoris genetically engineered bacterium integrates and expresses the cell wall adhesion protein gene in the Pichia pastoris GS115 genome, significantly enhancing the film-forming ability of the Pichia pastoris cells; then, multiple copies of the β-galactosidase surface display gene are integrated, and a constitutive promoter is used to control the display of the target protein β-galactosidase on the yeast surface. The Pichia pastoris genetically engineered bacterium with efficient surface display of enzymes based on adhesion proteins can carry out multiple batches of continuous catalysis and has better effects than enzyme surface display genetically engineered bacteria without integrated biofilm genes. After 6 batches, the relative conversion rate still remains at 78%, which has good prospects for industrial application in immobilized continuous catalysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a genetically engineered Pichia pastoris with high-efficiency surface display of enzymes based on adhesion proteins, a construction method and an application thereof. Background Art

[0002] At present, green and sustainable biomanufacturing is the core of achieving energy conservation and emission reduction, waste value-added and renewable resource reuse in large-scale industrial production. The development of cell immobilization technology is the main factor supporting cost reduction, efficiency improvement and continuous operation in the biomanufacturing process. Among them, biofilm immobilization technology mainly relies on the natural biological community formed by microbial cells wrapped in extracellular matrix (polysaccharides, proteins, amyloid proteins, lipids and extracellular DNA) spontaneously aggregating and adhering to the surface of the carrier. This natural microbial community exhibits high cell viability, cell regeneration ability and high tolerance to external stimuli. This immobilization strategy with great development potential has been used in processes such as continuous fermentation of Escherichia coli to produce amino acids, yeast fermentation of ethanol and Pichia pastoris to produce phytase. However, few studies have applied the biofilm immobilization strategy to continuous biocatalytic processes.

[0003] Traditional biocatalytic processes are severely limited by arduous enzyme purification procedures, enzyme instability, and difficulty recovering from the reaction medium. To address these drawbacks, whole-cell biocatalytic strategies have been proposed for enzyme display on microbial surfaces. Displayed enzymes coexist in a relatively stable cellular environment, effectively resisting environmental changes and maintaining high catalytic activity. This whole-cell approach also eliminates expensive enzyme purification steps and facilitates enzyme recovery and reuse, thereby improving the overall sustainability of biocatalytic applications. Furthermore, the displayed enzymes are directly exposed to the substrate, avoiding the inadequate cellular mass transfer caused by transmembrane transport of large substrates and excessive intracellular transport of substances. Nevertheless, the catalytic capacity of displayed enzymes is largely dependent on surface enzyme density. Strategies such as host modification, directed enzyme evolution, copy number optimization, and saturated enzyme loading at the single-cell level can improve display efficiency to achieve high displayed enzyme densities for efficient catalysis. However, continuous catalysis studies that exploit the high cell density of biofilm clusters to improve enzyme density have yet to be reported.

[0004] Pichia pastoris is a highly effective host for the industrial production of heterologous proteins and industrial enzymes, offering advantages such as high cell culture density, high-intensity expression, post-translationally modified protein processing, and freedom from endotoxin and viral contamination. However, its biofilm-forming ability is relatively weak, and actual fermentation processes have exposed problems such as unstable clusters and easy adsorption and shedding. Previous work has explored genes that promote biofilm formation using genetic engineering methods, making them suitable for continuous fixed fermentation. This has prompted those skilled in the art to comprehensively explore potential biofilm genes, explore the properties of Pichia pastoris adhesion carrier media, and expand its application in immobilized continuous catalysis by combining surface-displayed whole-cell catalysis methods, thereby shortening catalytic batches and recycling natural catalysts. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a genetically engineered Pichia pastoris strain that efficiently displays β-galactosidase on its surface based on adhesion protein. The genetically engineered Pichia pastoris strain can express the adhesion protein gene to enhance the ability of its cells to form biofilms, and has multiple copies of an expression cassette that integrates a fusion gene of the β-galactosidase gene LacA and the cell wall anchor protein gene Pir1p, thereby controlling the display of the target protein β-galactosidase on the surface of the Pichia pastoris. The strain is then applied to the immobilized continuous catalysis of lactose to produce oligomeric galactose, thereby achieving the purpose of shortening batch catalysis time and recycling the natural catalyst.

[0006] The technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered Pichia pastoris.

[0007] A further technical problem to be solved by the present invention is to provide the use of the above-mentioned genetically engineered Pichia pastoris in the immobilized continuous catalytic production of galacto-oligosaccharides from lactose.

[0008] In order to solve the first technical problem mentioned above, the present invention discloses a genetically engineered Pichia pastoris with efficient surface display of enzymes based on adhesion proteins, in which adhesion protein genes, β-galactosidase gene LacA and cell wall anchor protein gene Pir1p are heterologously expressed in the starting strain Pichia pastoris.

[0009] Wherein, the Pichia pastoris is Pichia pastoris GS115.

[0010] Wherein, the adhesion protein is a potential protein containing an adhesion functional protein domain (Flo11p domain, cellulose binding domain, GLEYA lectin-like binding domain or lectin Flo9 type III domain) and a mutant thereof with a deletion of the adhesion functional domain; the adhesion protein gene is any one of flo1, flo2, flo3, flo4, flo5-2, flo11, gcw16, gcw16ΔFlo11p and gcw16ΔCBD; the NCBI accession numbers of the genes flo1, flo4 and flo11 are PAS_chr1-4_0584, PAS_chr4_0151 and PAS_chr2-2_0482, respectively, and the nucleotide sequences of the genes flo2, flo3, flo5-2 and gcw16 are as shown in SEQ As shown in No. 1 to 4; the genes gcw16ΔFlo11p and gcw16ΔCBD are truncated genes of the gene gcw16. Compared with the gcw16-encoded protein, the proteins encoded by gcw16ΔFlo11p and gcw16ΔCBD lack the N-terminal 32 to 160 amino acids and 230 to 424 amino acids of the gcw16-encoded protein, respectively.

[0011] The nucleotide sequence of the β-galactosidase gene LacA is shown in SEQ No. 5; the nucleotide sequence of the cell wall anchor protein gene Pir1p is shown in SEQ No. 6.

[0012] The present invention further discloses a method for constructing the above-mentioned Pichia pastoris genetically engineered bacteria, wherein the expression cassette of the fusion gene Pir1p-LacA of the cell wall anchor protein gene Pir1p and the β-galactosidase gene LacA and the expression cassette of the adhesion protein gene are integrated into the genome of the Pichia pastoris using homologous recombination technology.

[0013] Wherein, a single copy or multiple copies of the expression cassette of the fusion gene Pir1p-LacA are integrated into the genome of the Pichia pastoris; the copy number of the multiple copies is preferably 2 to 4.

[0014] Wherein, the expression cassette of the fusion gene Pir1p-LacA is expressed under the control of a constitutive promoter, and the constitutive promoter is preferably a glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP).

[0015] Specifically, the construction method of the Pichia pastoris genetically engineered bacteria is as follows:

[0016] (1) Using overlapping PCR technology (overlap PCR), an expression cassette of the fusion gene Pir1p-LacA was constructed (using the recombinant plasmid pPIC9k-pir1p-lacA in patent CN114606151A as a template, and the fusion gene Pir1p-LacA fragment was obtained by PCR amplification);

[0017] (2) using CRISPR / Cas9 gene editing technology to integrate the expression cassette of the fusion gene Pir1p-LacA obtained in step (1) into the Pichia pastoris genome to obtain a genetically engineered Pichia pastoris strain displaying β-galactosidase on its surface;

[0018] (3) Insert the adhesion gene into plasmid pGAPZαA to construct a recombinant plasmid;

[0019] (4) using the recombinant plasmid obtained in step (3) as a template, obtaining the expression cassette of the adhesion gene by PCR amplification;

[0020] (5) Using CRISPR / Cas9 gene editing technology, the expression cassette of the adhesion gene obtained in step (4) is integrated into the genome of the Pichia pastoris genetically engineered bacteria displaying β-galactosidase on the surface obtained in step (2), thereby obtaining the Pichia pastoris genetically engineered bacteria displaying β-galactosidase on the surface based on the adhesion protein.

[0021] In order to solve the second technical problem mentioned above, the present invention further discloses the use of the genetically engineered Pichia pastoris in the continuous immobilized catalytic production of galacto-oligosaccharides from lactose.

[0022] Specifically, the genetically engineered Pichia pastoris is inoculated into a YPD liquid culture medium and cultured overnight to obtain a seed solution, which is then inoculated into a YPD culture medium containing an immobilized carrier for immobilized fermentation. The supernatant fermentation solution is discarded, and lactose is used as a substrate to batch catalyze the production of oligomeric galactose from lactose.

[0023] The seed solution is inoculated into a YPD liquid medium containing an immobilized carrier so that the initial OD value of the Pichia pastoris genetically engineered bacteria is 600 It is 1.0~5.0.

[0024] Among them, the specific conditions of the immobilized fermentation are: immobilized fermentation culture is carried out at 28-30°C and 220-250rpm for 90-150h; the batch catalysis of lactose to produce oligomeric galactose has a catalytic time of 12-24h for each batch and a catalytic temperature of 45-65°C.

[0025] Among them, the immobilization carrier includes cotton fiber, polypropylene, polyethylene, loofah capsule, polyester fiber, bacterial cellulose membrane, bamboo fiber, central control fiber membrane, etc.

[0026] The YPD liquid culture medium comprises the following components: 10-15 g / L yeast powder, 15-25 g / L peptone and 15-25 g / L glucose.

[0027] The genetically engineered Pichia pastoris of the present invention effectively improves the problems of weak film-forming ability of Pichia pastoris, difficulty in adsorbing carriers, poor continuous effect, etc., thereby realizing the application of the genetically engineered Pichia pastoris in biofilm immobilized continuous catalysis.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] (1) The Pichia pastoris genetically engineered bacteria with efficient surface display of enzymes based on adhesion proteins disclosed in the present invention integrate the adhesion protein gene into the Pichia pastoris genome for expression. The Pichia pastoris genetically engineered bacteria have enhanced cell biofilm formation ability during static and dynamic culture processes, stronger adsorption capacity in immobilized fermentation culture, and increased the amount of adherent cells in immobilized fermentation. Compared with the original strain GS115, the dry weight of adherent cells of the Pichia pastoris genetically engineered bacteria increased by approximately 17.0%.

[0030] (2) The genetically engineered Pichia pastoris bacteria disclosed in the present invention utilizes a constitutive promoter to regulate the expression of the fusion gene Pir1p-LacA, which is a cell wall anchor protein gene Pir1p and the β-galactosidase gene LacA. This allows the exogenous fusion gene Pir1p-LacA to be expressed as the Pichia pastoris cells grow, and the β-galactosidase is displayed on the surface of the Pichia pastoris cells. There is no need to exogenously add cytotoxic methanol to induce the expression of the fusion gene Pir1p-LacA.

[0031] (3) The genetically engineered Pichia pastoris disclosed in the present invention integrates multiple copies of the expression cassette of the fusion gene Pir1p-LacA, which is a cell wall anchor protein gene Pir1p and the β-galactosidase gene LacA, into the Pichia pastoris genome. The cellular enzyme activity of β-galactosidase gradually increases with the increase in the copy number of the expression cassette. In the catalytic process using lactose as a substrate, the catalytic effect of the genetically engineered Pichia pastoris with two copies of the expression cassette integrated has reached saturation.

[0032] (4) The genetically engineered Pichia pastoris disclosed in the present invention has both film-forming adsorption ability and enzyme efficient display ability, and can carry out multiple batches of immobilized continuous catalysis of lactose to produce oligomeric galactose, and the catalytic effect can maintain a certain stability. After 6 batches of catalysis, the relative conversion rate of lactose still remains at 78%, which has good industrial prospects in continuous catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0034] Figure 1 The agarose gel electrophoresis diagram of the recombinant plasmid and Pichia pastoris transformants constructed in Example 1. Figure 1 Figure a in the middle is the electrophoresis diagram of adhesion gene fragments, where lanes 1 to 7 are gcw16, flo1, flo2, flo3, flo4, flo5-2, and flo11 gene fragments, respectively, and lane 8 is the DNA marker of DL5000; Figure 1 Figure b is the electrophoresis diagram of the vector fragment, where lane 1 is the linearized fragment of the pGAPZαA vector plasmid and lane 2 is the DL5000 DNA marker.

[0035] Figure 2 The Flo11p-binding domain and CBD-binding domain of the adhesion protein encoded by gcw16.

[0036] Figure 3 This is the electrophoresis diagram of the truncated gene fragment of gcw16 and the Pichia pastoris transformant obtained in Example 2; Figure 3 Figure a in the middle shows the electrophoresis of the truncated gene and vector fragments of gcw16, where lanes 1 and 2 are the two gene fragments of gcw16ΔFlo11p, respectively; lanes 3 and 4 are the two gene fragments of gcw16ΔCBD, respectively; lane 5 is the pGAPZαA vector fragment; lane 6 is the DL5000 DNA marker; Figure 3 Figure b is the PCR identification electrophoresis diagram of the recombinant yeast transformants integrated with the truncated gene of gcw16, where lane 1 is the DL10000 DNA marker; lane 2 is the original Pichia pastoris control band; lanes 3 and 4 are positive transformants integrated with the gene gcw16ΔFlo11p; lanes 5 and 6 are positive transformants integrated with the gene gcw16ΔCBD.

[0037] Figure 4 Statistical graphs of biofilm formation of the original Pichia pastoris GS115 and genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 3 and staining images of the bottom of the well plate.

[0038] Figure 5 Graphs showing the plate invasion and plate growth results of the original Pichia pastoris GS115 and genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 4.

[0039] Figure 6This is a statistical graph of the biomass adsorbed on the bottom of the well plate by the original Pichia pastoris GS115 and the genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 5.

[0040] Figure 7 The graph shows the OD600 of free cells and the biomass adsorbed on the carrier during the cotton fiber immobilization fermentation process of the original bacteria (GS115) and the genome-integrated recombinant engineered bacteria (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 6.

[0041] Figure 8 This is the nucleic acid electrophoresis diagram of the enzyme display expression cassette in Example 7; wherein, lane 1 is the promoter pGAP fragment; lane 2 is the terminator AOXTT fragment; lane 3 is the fusion gene Pir1p-LacA gene fragment; lane 4 is the expression cassette pGAP-Pir1p-LacA-AOXTT fragment; lane 5 is the DL5000 DNA marker.

[0042] Figure 9 The changes in cellular enzyme activities of the genetically engineered Pichia pastoris strains +1*Pir1p-LacA, +2*Pir1p-LacA, +3*Pir1p-LacA, and +4*Pir1p-LacA during 120 h of fermentation in Example 7 are shown.

[0043] Figure 10 The cellular enzyme activity of the multi-copy enzyme display genetically engineered bacteria after integration of the adhesion protein gene in Example 8 and the yield of galacto-oligosaccharides produced by single-batch catalysis of lactose, wherein Figure a shows the cellular enzyme activity and Figure b shows the yield of galacto-oligosaccharides.

[0044] Figure 11 The results show that the high-efficiency enzyme based on adhesion proteins in Example 9 can produce galacto-oligosaccharides from lactose in continuous immobilized batches by genetically engineered bacteria.

[0045] Figure 12 The relative conversion rate of lactose conversion catalyzed by the genetically engineered bacteria in continuous immobilized batches is shown for the high-efficiency enzyme based on adhesion proteins in Example 9. DETAILED DESCRIPTION

[0046] The present invention is further described below with reference to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0047] The plasmids, endonucleases, high-fidelity enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercial products, and specific procedures were performed according to the kit instructions. All other experimental consumables, unless otherwise specified, were commercially available. The following experimental procedures, including preparation of competent cells, colony PCR, nucleic acid agarose gel electrophoresis, electrotransformation, and yeast genome extraction and storage, were performed according to conventional methods.

[0048] The Pichia pastoris starting strain used in the following examples was GS115.

[0049] The genomic scarless integration expression system used in the following examples is a CRISPR-Cas9 gene editing plasmid, which was donated by Professor Zhou Yongjin of the Synthetic Microbiology Research Group of the Dalian Institute of Chemical Physics, Chinese Academy of Sciences.

[0050] The sequencing of plasmids and DNA products in the following examples was commissioned to Anhui General Biotechnology Co., Ltd.

[0051] In the following examples, the concentrations of lactose, galacto-oligosaccharides, glucose, and galactose in the catalyst solution were determined as follows: 1 mL of the catalyst solution was boiled at 100°C for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was diluted a predetermined number of times before filtering through a 0.22 μm filter membrane. The sugar concentrations of the various components in the catalyst system were analyzed using high-performance liquid chromatography (HPLC). HPLC detection methods included a differential refractive index detector (DRD); a BioRad HPX-87H (9 μm, 300 mm × 7.8 mm) column, a column temperature of 60°C, a 5 mmol / L aqueous H₂SO₄ mobile phase at a flow rate of 0.4 mL / min, and a 5 μL injection volume.

[0052] The formula of the YPD liquid medium described in the following examples is 10-15 g / L yeast powder, 15-25 g / L peptone and 15-25 g / L glucose, and the formula of the YPD solid medium is 10-15 g / L yeast powder, 15-25 g / L peptone, 15-25 g / L glucose and 15-25 g / L agar.

[0053] Example 1 Construction of a genetically engineered Pichia pastoris strain based on adhesion proteins

[0054] Multiple recombinant expression plasmids carrying adhesion protein genes flo1, flo2, flo3, flo4, flo5-2, flo11, and gcw16 were constructed respectively; then, using Pichia pastoris GS115 as the starting strain, the homologous recombination system was used to integrate the entire plasmid genome into the promoter pGAP site for expression. The primer sequences involved are shown in Table 1. The specific construction steps are as follows:

[0055] (1) Amplification of target gene fragments: Using the Pichia pastoris GS115 genome as a template, the target gene respective primers were used to amplify the gene fragments gcw16, flo1, flo2, flo3, flo4, flo5-2, and flo11, and then recovered by gel electrophoresis. The agarose gel electrophoresis diagram of the above gene fragments is shown in Figure 2. Figure 1 As shown in a.

[0056] (2) Amplification of vector fragments: PCR amplification was performed using the original plasmid pGAPZαA as a template and GAPαA-F / GAPαA-R as primers. The amplified product was digested with restriction endonuclease Dpn I to eliminate the excess circular pGAPZαA plasmid and obtain a fragment of the vector pGAPZαA. The agarose gel electrophoresis diagram is shown in FIG. Figure 1 As shown in b.

[0057] (3) Construction of linearized recombinant plasmid fragments: The adhesion gene fragments obtained in step (1) were cloned and connected with the vector fragments obtained in step (2) and transformed into Escherichia coli DH5α competent cells, which were then plated on LB plates containing bleomycin. Single colonies were picked to extract plasmids and sequenced. The correct sequencing results were pGAPZαA plasmids with the adhesion protein gene successfully inserted. They were named pGAPZαA-flo1, pGAPZαA-flo2, pGAPZαA-flo3, pGAPZαA-flo4, pGAPZαA-flo5-2, pGAPZαA-flo11 and pGAPZαA-gcw16, respectively. The above recombinant plasmids were cut into linearized recombinant plasmids using restriction endonuclease ArVII, and the linearized recombinant plasmids were recovered and purified by gel recovery.

[0058] (4) Construction of genetically engineered Pichia pastoris strains: Using Pichia pastoris GS115 as the starting strain, the linearized plasmids obtained in step (3) were electroporated into Pichia pastoris GS115 competent cells, which were then plated onto YPD plates (containing 100 mM bleomycin) and cultured at 30°C and 250 rpm until single colonies appeared. Colony PCR was performed on the single colonies grown on the plates, and positive transformants were screened and sequenced. In this example, a total of seven Pichia pastoris genetically engineered strains based on adhesion proteins were constructed, namely +flo1, +flo2, +flo3, +flo4, +flo5-2, +flo11, and +gcw16.

[0059] Table 1 Primer sequences required for constructing recombinant plasmids

[0060]

[0061] Example 2 Construction of a genetically engineered Pichia pastoris strain based on the gcw16 truncated gene

[0062] Figure 2 The Flo11p binding domain and CBD binding domain of the adhesion protein encoded by gcw16. As shown in the figure, the protein encoded by the adhesion protein gene gcw16 contains two potential adhesion protein domains, namely the Flo11p binding domain and the CBD binding domain.

[0063] Recombinant expression plasmids for the truncated gcw16 gene gcw16ΔFlo11p (the protein encoded by this gene lacks the Flo11p binding domain compared to the protein encoded by gcw16) and gcw16ΔCBD (the protein encoded by this gene lacks the CBD binding domain compared to the protein encoded by gcw16) were constructed respectively; then, using Pichia pastoris GS115 as the starting strain, the homologous recombination system was used to integrate the entire plasmid genome into the promoter pGAP site for expression. The primer sequences involved are shown in Table 2. The specific construction steps are as follows:

[0064] (1) Amplification of target gene fragments: Using the Pichia pastoris GS115 genome as a template, primers gcw16-F / gcw16ΔFlo11p-R and gcw16ΔFlo11p-F / gcw16-R were used to amplify the double fragments of the target gene gcw16ΔFlo11p, and primers gcw16-F / gcw16ΔCBD-R and gcw16ΔCBD-F / gcw16-R were used to amplify the double fragments of the target gene gcw16ΔCBD, and the fragments were recovered by gel electrophoresis. The agarose gel electrophoresis diagram of the above target gene fragments is shown in Figure 2. Figure 3 As shown in a.

[0065] (2) Amplification of plasmid fragments: PCR amplification was performed using the original pGAPZαA as a template and GAPαA-F / GAPαA-R as primers. The amplified product was digested with restriction endonuclease Dpn I to eliminate the excess circular pGAPαA plasmid and obtain a fragment of the vector pGAPZαA. The agarose gel electrophoresis pattern is shown in Figure 2. Figure 3 As shown in a.

[0066] (3) Construction of linearized recombinant plasmid fragments: The gcw16ΔFlo11p and gcw16ΔCBD gene fragments obtained in step (1) were cloned and ligated with the vector fragment obtained in step (2) and transformed into Escherichia coli DH5α competent cells, which were then plated on LB plates containing bleomycin. Single colonies were picked to extract the plasmids and sequenced. The plasmids with correct sequencing were the pGAPZαA plasmids that had successfully inserted the target gene. They were named pGAPZαA-gcw16ΔFlo11p and pGAPZαA-gcw16ΔCBD, respectively. The above recombinant plasmids were digested with restriction endonuclease ArVII to form linearized recombinant plasmids, and the linearized recombinant plasmids were recovered and purified by gel recovery.

[0067] (4) Construction of genetically engineered Pichia pastoris strains: Using Pichia pastoris GS115 as the starting strain, the linearized plasmids obtained in step (3) were electroporated into Pichia pastoris GS115 competent cells, which were then plated on YPD plates (100 mM bleomycin) and cultured at 30°C and 250 rpm until single colonies appeared. The single colonies grown on the plates were verified by colony PCR, and positive transformants were screened and sequenced. Figure 3 As shown in b, relative to the original Pichia pastoris band (about 1100 bp), gcw16ΔFlo11p and gcw16ΔCBD were successfully integrated into its genome, respectively, to obtain Pichia pastoris genetically engineered strains +gcw16ΔFlo11p and +gcw16ΔCBD based on the gcw16 truncated gene.

[0068] Table 2 Primer sequences required for the construction of recombinant plasmids of gcw16 truncated genes

[0069]

[0070] Example 3 Crystal violet staining experiment

[0071] (1) The original Pichia pastoris GS115 and all the Pichia pastoris genetically engineered bacteria integrated with adhesion genes in Examples 1 and 2 were inoculated into 5 mL of sterilized YPD liquid medium and cultured overnight for activation;

[0072] (2) The activated bacterial solution in step (1) was inoculated into 100 mL of YPD liquid medium at a 1% inoculum volume, and cultured at 30°C and 250 rpm until the bacterial solution OD 600 The value is between 0.8 and 1.2;

[0073] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterilized YPD liquid medium so that the OD of the diluted bacterial solution is 600 is 0.01;

[0074] (4) 200 μL of the diluted bacterial solution was added to a 96-well plate, and YPD liquid medium was used as a control. The plates were cultured at 30°C for 2, 3, and 5 days.

[0075] (5) Pour out the bacterial solution in the 96-well plate, take 200 μL of 0.01 M PBS buffer to wash the 96-well plate 2–3 times, and pat dry;

[0076] (6) Add 200 μL of 0.1% w / v crystal violet aqueous solution (1 g / L) to the 96-well plate prepared in step (5) and stain for 10–20 min. Rinse 2–3 times with 0.01 M PBS buffer and pat dry.

[0077] (7) Take 200 μL of glacial acetic acid and add it to the 96-well plate in step (5) to dissolve the biofilm. Gently shake for 40 minutes and measure the OD 560 (OD 560 The values reflect the amount of biofilm formation) and the average value was taken.

[0078] Biofilm formation amount (OD 560 ) The results are shown in Table 3. The data in Table 3 indicate that the integration of adhesion protein genes is beneficial to the biofilm formation of Pichia pastoris, among which the adhesion protein gene gcw16 and its truncated genes gcw16ΔFlo11p and gcw16ΔCBD significantly improved its film-forming ability. Figure 4 The following are statistical graphs of the biofilm formation of the original Pichia pastoris (GS115) and genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) cultured for 5 days in this example, as well as staining images of the bottom of the well plate. It can be seen from the figure that the gcw16ΔFlo11p gene, which is a truncated version of the gcw16 gene, is more conducive to the formation of biofilm structures by Pichia pastoris on the well plate (made of polyethylene).

[0079] Table 3 The amount of biofilm formed by genetically engineered Pichia pastoris with integrated adhesion protein genes

[0080]

[0081] Example 4 Flat plate invasion and flat plate growth experiments

[0082] (1) The original Pichia pastoris GS115, +gcw16 constructed in Example 1, +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were inoculated into 5 mL of sterilized YPD liquid medium and cultured overnight for activation;

[0083] (2) The activated bacterial solution in step (1) was inoculated into 100 mL of YPD liquid medium at a 1% inoculum volume, and cultured at 30°C and 250 rpm until the bacterial solution OD 600 The value is between 0.8 and 1.2;

[0084] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterilized YPD liquid medium so that the OD of the diluted bacterial solution is 600 1, 0.1, 0.01 and 0.001;

[0085] (4) Take 2 μL of the diluted bacterial solution and drop it vertically on the YPD solid culture medium plate. After culturing at 30°C for 1 day, record the growth status of the strains at each concentration on the plate. At the same time, take the OD 60010 μL of bacterial solution with a concentration of 1 was dropped onto a YPD solid culture medium plate. After culturing at 30°C for 5 days, the invasion ability of each strain on the plate was recorded.

[0086] The experimental results are as follows Figure 5 As shown, compared with the original Pichia pastoris GS115, the plate invasion ability of the Pichia pastoris genetically engineered bacteria with integrated adhesion protein genes was not enhanced, and the integrated expression of the gene did not inhibit the growth of Pichia pastoris itself.

[0087] Example 5: Well plate adsorption cell counting experiment under static culture

[0088] (1) The original Pichia pastoris GS115, +gcw16 constructed in Example 1, +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were inoculated into 5 mL of sterilized YPD liquid medium and cultured overnight for activation;

[0089] (2) The activated bacterial solution in step (1) was inoculated into 100 mL of YPD liquid medium at a 1% inoculum volume, and cultured at 30°C and 250 rpm until the bacterial solution OD 600 The value is between 0.8 and 1.2;

[0090] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterilized YPD liquid medium so that the OD of the diluted bacterial solution is 600 is 0.01;

[0091] (4) 200 μL of the diluted bacterial solution was added to a 96-well plate, and YPD liquid medium was used as a control. The plates were cultured at 30°C for 3 days.

[0092] (5) Pour out the bacterial solution in the 96-well plate in a clean bench and wash 2–3 times with 200 μL of PBS buffer.

[0093] (6) Subsequently, 200 μL of 0.01 M sterile PBS buffer (containing 0.01 M proteinase K) was used to desorb the adherent cells in the 96-well plate for 30 min;

[0094] (7) Finally, the desorbed cell solution was gradiently diluted 100-fold, 1000-fold, and 10,000-fold, and 100 μL was spread on YPD solid culture medium plates. After incubation at 30°C for 3 days, the plates were counted. Three parallels were set up for each group and the average value was taken.

[0095] The experimental results are as follows Figure 6 As shown in the figure, compared with the original Pichia pastoris GS115, the number of attached cells of the genetically modified Pichia pastoris increased significantly, among which the number of attached cells of the genetically modified Pichia pastoris overexpressing the truncated gene gcw16ΔFlo11p was 8.4×104 CFU / μL is 2 orders of magnitude higher than that of the original Pichia pastoris GS115.

[0096] Example 6 Adsorption Effect of Pichia Pastoris Genetically Engineered Bacteria During Dynamic Immobilization Fermentation

[0097] (1) The original Pichia pastoris GS115, +gcw16 constructed in Example 1, +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were inoculated into 5 mL of sterilized YPD liquid medium and cultured overnight for activation;

[0098] (2) The activated bacterial solution in step (1) was inoculated into 100 mL of YPD liquid culture medium at a 1% inoculum volume, and cultured at 30°C and 250 rpm for 24 h;

[0099] (3) Then the bacterial solution OD in step (2) 600 The pH value was adjusted to 1.0 and transferred to 50 mL of YPD liquid medium containing 2 g of cotton fiber carrier for immobilized fermentation culture at 30°C and 250 rpm. The free cell density in the fermentation supernatant was recorded every 12 h. At the same time, the cotton fiber medium with adsorbed microbial cells was taken out on the 1st, 2nd, and 3rd day, dried at 65°C, weighed, and the dry weight of the microbial cells adsorbed on the medium was obtained by subtracting the initial weight of 2 g of cotton fiber carrier.

[0100] The experimental results are as follows Figure 7 As shown, the +gcw16ΔFlo11p strain, which exhibited the best film-forming effect in static culture, showed a consistent trend in the free cell content of its dynamic fermentation supernatant with that of the original Pichia pastoris GS115, both exhibiting a high number of planktonic cells. The fermentation supernatants of the +gcw16 and +gcw16ΔCBD strains did not contain excessive free cells, and the dry weight of microbial cells adsorbed on the cotton fiber medium was significantly higher than that of the original Pichia pastoris GS115 and +gcw16ΔFlo11p strains. After three days of culture, the amount of cells adhered to the carrier increased by approximately 17.0% for +gcw16 and +gcw16ΔCBD compared to the original Pichia pastoris GS115. These results demonstrate that the Pichia pastoris genetically engineered strains based on adhesion proteins constructed by the present invention can also be used in dynamic immobilized fermentation cultures, and that the adsorption effect in immobilized fermentation is more robust and less susceptible to detachment.

[0101] Example 7 Construction of a genetically engineered Pichia pastoris strain displaying β-galactosidase on its surface

[0102] The constitutive promoter pGAP of Pichia pastoris was used to construct an expression cassette of the fusion gene Pir1p-LacA (derived from patent CN114606151A), which is a fusion gene of the anchor protein gene Pir1p and the β-galactosidase gene LacA. The expression cassette was integrated into the Pichia pastoris genome using homologous recombination technology and a gene editing system (due to the need for multiple rounds of genome editing, the gene editing system designed in this embodiment is a CRISPR / Cas9 system). This constructed a fermentation system for enzyme display expression coupled with cell growth, effectively avoiding the cytotoxicity and environmental pollution hazards caused by the inducer methanol. The primers involved are shown in Table 4, and the specific construction steps are as follows:

[0103] (1) Using the vector pGAPZαA as a template, the promoter pGAP and terminator AOXTT fragments were obtained by PCR amplification using primers pGAP-F / pGAP-R and AOXTT-F / AOXTT-R; using the recombinant plasmid pPIC9k-pir1p-lacA in patent CN114606151A as a template, the fusion gene Pir1p-LacA fragment was amplified by PCR using primers Pir1p-F / LacA-R; using the above three fragments as templates, the expression cassette pGAP-Pir1p-LacA-AOXTT fragment was amplified by overlapping PCR technology (overlap PCR) using primers pGAP-F / AOXTT-R, and then gel recovery and purification were performed.

[0104] (2) Using the Pichia pastoris GS115 genome as a template, primers PNSI-2-UF / PNSI-2-UR and PNSI-2-DF / PNSI-2-DR were used to amplify the upper and lower homologous arms I-2-Up and I-2-Down of the site (PNSI-2); finally, the upper and lower homologous arm fragments were overlapped with the gene expression cassette constructed in step (1) using primers PNSI-2-UF / PNSI-2-DR to obtain the Donor DNA fragment, which was then recovered and purified by gel recovery.

[0105] (3) 5 μg of the donor DNA fragment purified in step (2) and 1 μg of the CRISPR / Cas9 plasmid containing the PNSI-2 site were electroporated into Pichia pastoris competent cells, which were then plated on YPD solid culture plates (containing 100 mM bleomycin) and cultured at 30°C and 250 rpm until single colonies appeared. The single colonies grown on the plates were verified by colony PCR, positive transformants were screened and sequenced, and multiple rounds of passage were performed to lose the tool plasmid. Finally, the Pichia pastoris genetically engineered strain +1*Pir1p-LacA was successfully constructed.

[0106] (4) Repeat steps (2) and (3) to sequentially integrate the expression cassette pGAP-Pir1p-LacA-AOXTT into the PNSⅠ-3, PNSⅡ-4, and PNSⅢ-5 sites of the Pichia pastoris genome. Finally, 2-copy, 3-copy, and 4-copy enzyme-displaying Pichia pastoris genetic engineering strains +2*Pir1p-LacA, +3*Pir1p-LacA, and +4*Pir1p-LacA were successfully constructed.

[0107] The nucleotide sequences of the Pichia pastoris genomic sites PNSI-2, PNSⅠ-3, PNSⅡ-4 and PNSⅢ-5 are shown in SEQ No. 7 to 10 respectively.

[0108] Figure 8 This is the nucleic acid electrophoresis diagram of the enzyme display expression cassette in this example.

[0109] The multi-copy enzyme-displaying genetically engineered bacteria constructed above were fermented and assayed for enzyme activity. The specific fermentation method was as follows: single clones of the Pichia pastoris genetically engineered strain +1*Pir1p-LacA, +2*Pir1p-LacA, +3*Pir1p-LacA, and +4*Pir1p-LacA were transferred to 5 mL of YPD liquid medium and cultured overnight for activation; then, at a 1% inoculum volume, the cells were transferred to 50 mL of YPD liquid medium and cultured continuously at 30°C and 250 rpm; and the cellular enzyme activity in the fermentation broth was assayed every 24 hours.

[0110] The cellular enzyme activity was detected according to the method in patent CN114606151A, as follows: o-Nitrophenyl-β-D-galactoside (oNPG) was used as the substrate, an appropriate amount of enzyme-displaying genetically engineered bacterial cells was added, and the reaction was carried out at a catalytic temperature of 50°C for 10 minutes. The enzyme activity unit 1U was defined as the amount of cells required to convert 1 nmol of oNPG per minute.

[0111] The results of cell enzyme activity test were as follows Figure 9 As shown, the multi-copy enzyme display genetic engineering strain constructed above can secrete and display β-galactosidase on the cell wall as the yeast cells grow without the addition of inducers; moreover, as the copy number of the enzyme display gene expression cassette increases, the activity of the displayed enzyme gradually increases.

[0112] Table 4 Primer sequences required for constructing Pichia pastoris genetically engineered enzyme displaying β-galactosidase on the surface

[0113]

[0114]

[0115] Example 8 Construction of Pichia pastoris genetically engineered bacteria with high-efficiency surface display of β-galactosidase based on adhesion proteins and verification of its single-batch catalytic effect

[0116] The construction of Pichia pastoris genetically engineered bacteria with high-efficiency surface display of β-galactosidase based on adhesion proteins is as follows:

[0117] (1) Using the recombinant plasmid pGAPZαA-gcw16ΔCBD in Example 2 as a template, the corresponding expression cassette pGAP-gcw16ΔCBD-AOXTT was amplified using primers pGAP-F / pGAP-R, and the fragments were recovered and purified on gel. Then, using the Pichia pastoris GS115 genome as a template, the upper and lower homology arms IV-2-Up and IV-2-Down of the site (PNSIV-2) were amplified using primers PNSIV-2-UF / PNSIV-2-UR and PNSIV-2-DF / PNSIV-2-DR. Finally, the upper and lower homology arm fragments were overlapped with the gene expression cassette using primers PNSIV-2-UF / PNSIV-2-DR to obtain the Donor DNA fragment, which was then recovered and purified on gel.

[0118] (2) 5 μg of the purified Donor DNA fragment from step (1) and 1 μg of the CRISPR / Cas9 plasmid containing the PNSⅣ-2 site were electroporated into the competent cells of the multi-copy enzyme display Pichia pastoris genetic engineering bacteria in Example 7, and then spread on a YPD plate (containing 100 mM bleomycin) and cultured at 30°C and 250 rpm until a single colony appeared. The single colony grown on the plate was verified by colony PCR, and the positive transformants were screened and sequenced. Finally, the Pichia pastoris genetic engineering bacteria +gcw16ΔCBD,1*Pir1p-LacA, +gcw16ΔCBD,2*Pir1p-LacA, +gcw16ΔCBD,3*Pir1p-LacA, +gcw16ΔCBD,4*Pir1p-LacA based on adhesion protein were successfully constructed.

[0119] The nucleotide sequence of the Pichia pastoris genomic site PNSⅣ-2 is shown in SEQ No.11.

[0120] The primer sequences required in the above construction process are shown in Table 5.

[0121] The multi-copy enzyme-displaying recombinant bacteria constructed above, based on the film-forming gene, were fermented and assayed for enzyme activity. The specific fermentation method was as follows: a single clone of the genetically engineered bacteria was transferred to 5 mL of YPD liquid medium for overnight activation. The inoculum was then transferred to 50 mL of YPD liquid medium at a 1% inoculum volume and cultured at 30°C and 250 rpm. The enzyme activity in the fermentation broth was recorded after 120 hours.

[0122] The multi-copy enzyme-displaying recombinant bacteria constructed above, based on the film-forming gene, were fermented and then subjected to single-batch catalysis using lactose as a substrate. The reaction conditions were 400 g / L lactose, 50°C, and 24 hours. The galacto-oligosaccharide content in the catalytic solution was determined by HPLC.

[0123] The results of cell enzyme activity and polygalactose production were as follows Figure 10 As shown. Figure 10 As can be seen in a, after the truncated gene gcw16ΔCBD expressing the adhesion protein was integrated into the recombinant strains with different copy numbers of the enzyme display gene, the cellular enzyme activity of β-galactosidase was slightly improved compared with the recombinant strains without gcw16ΔCBD integration. This may be because the GPI binding site contained in the adhesion protein encoded by gcw16ΔCBD is targeted to the cell wall, causing the cell wall structure of the recombinant strain to change, resulting in an increase in the anchored enzyme protein. Figure 10 As shown in Figure b, in a single-batch catalytic process using lactose as a substrate, the integration of gcw16ΔCBD into a genetically engineered strain harboring a single copy of the integrase display gene significantly increased galacto-oligosaccharide (GOS) production by 17.8% compared to the recombinant strain without gcw16ΔCBD integration. However, the expected increase in GOS production with increasing enzyme display gene copy number did not occur. At 2 enzyme display gene copies, the recombinant strain's catalytic efficiency for GOS production from lactose reached saturation.

[0124] Table 5 Primer sequences required for constructing genetically engineered strains in Example 8

[0125]

[0126] Example 9: Application of Pichia pastoris genetically engineered bacteria with high-efficiency surface display of β-galactosidase based on adhesion proteins in the immobilized continuous batch catalysis of lactose to produce galacto-oligosaccharides

[0127] To investigate the catalytic efficiency of an enzyme-displaying genetically engineered bacteria based on adhesion proteins in immobilized continuous batch catalysis, this example used a Pichia pastoris genetically engineered bacteria +gcw16ΔCBD,2*Pir1p-LacA that integrates two copies of the enzyme display gene and a single copy of the gcw16ΔCBD gene as the target strain, and +2*Pir1p-LacA as the control strain. The target strain and the control strain were cultured for 120 h according to the immobilized fermentation culture steps in Example 6 to allow sufficient adsorption of the strains on the cotton fiber support. The immobilized cell media were then placed in a catalytic system (400 g / L lactose, 100 mM sodium citrate buffer, pH 5.5, 50° C.) for continuous catalysis. The catalytic time for each batch was 24 h, and the catalytic solution was replaced every 24 h. The method for detecting the sugar component content in the catalytic system and catalytic solution was the same as in Example 8.

[0128]

[0129] Where M(GOS3) and M(GOS4) refer to the concentrations of galacto-oligosaccharides with a degree of polymerization of 3 and 4 at a certain time during the catalytic reaction (g / L); M(Lac0) refers to the initial concentration of lactose added to the catalytic system (g / L); GOS conversion rate (+gcw16ΔCBD,2*Pir1p-LacA,batch 1) refers to the GOS conversion rate of batch 1 in the continuous batches of lactose-to-oligosaccharides catalyzed by the strain +gcw16ΔCBD,2*Pir1p-LacA. GOS conversion rate (batch n) refers to the GOS conversion rate of batch n in the continuous batches of lactose-to-oligosaccharides catalyzed by the strain +gcw16ΔCBD,2*Pir1p-LacA or +gcw16ΔCBD.

[0130] The yield results of 6 consecutive batches of catalytic lactose production of oligosaccharides are as follows Figure 11 As shown, unlike Example 8, the yield of lactose-catalyzed product galacto-oligosaccharides (GOS) of the 2-copy enzyme display strain + gcw16ΔCBD,2*Pir1p-LacA integrated with the gcw16ΔCBD gene was significantly higher than that of the 2-copy enzyme display strain + 2*Pir1p-LacA without the gcw16ΔCBD gene, indicating that the integration of the gcw16ΔCBD gene is more conducive to cell adhesion to the cotton fiber carrier, thereby increasing the density of the enzyme and enhancing the catalytic effect. Figure 12 The relative conversion rate of lactose conversion catalyzed by +gcw16ΔCBD,2*Pir1p-LacA in continuous immobilized batches. In terms of relative conversion rate, after 6 batches of catalysis, the relative conversion rate of the two-copy enzyme display strain integrated with the gcw16ΔCBD gene remained at around 76%.

[0131] The present invention provides a genetically engineered Pichia pastoris strain for enzyme surface display based on adhesion proteins, and the concepts and methods for its construction. There are numerous methods and approaches for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this example may be implemented using existing technologies.

Claims

1. A genetically engineered Pichia pastoris with efficient surface display of enzymes based on adhesion proteins, characterized in that: An adhesion protein gene is homologously expressed, a β-galactosidase gene LacA is heterologously expressed, and a cell wall anchor protein gene Pir1p is heterologously expressed in a starting strain Pichia pastoris; the adhesion protein gene is gcw16 or gcw16ΔCBD; the nucleotide sequence of gcw16 is shown in SEQ No. 4; the nucleotide sequence of the β-galactosidase gene LacA is shown in SEQ No. 5; the nucleotide sequence of the cell wall anchor protein gene Pir1p is shown in SEQ No. 6; the gcw16ΔCBD is a truncated gene of gcw16, and compared with the protein encoded by gcw16, the protein encoded by gcw16ΔCBD lacks 230 to 424 amino acids at the N-terminus of the protein encoded by gcw16.

2. The genetically engineered Pichia pastoris according to claim 1, characterized in that The Pichia pastoris is Pichiapastoris GS115.

3. The method for constructing the genetically engineered Pichia pastoris according to claim 1 or 2, characterized in that: The expression cassette of the fusion gene Pir1p-LacA of the cell wall anchor protein gene Pir1p and the β-galactosidase gene LacA and the expression cassette of the adhesion protein gene are integrated into the genome of the Pichia pastoris using CRISPR / Cas9 gene editing technology.

4. The construction method according to claim 3, characterized in that A single copy or multiple copies of the expression cassette of the fusion gene Pir1p-LacA are integrated into the genome of the Pichia pastoris.

5. The construction method according to claim 3, characterized in that The expression cassette of the fusion gene Pir1p-LacA and the expression cassette of the adhesion protein gene are both expressed under the control of a constitutive promoter.

6. Use of the genetically engineered Pichia pastoris according to claim 1 or 2 in the continuous immobilized catalytic production of galacto-oligosaccharides from lactose.

7. The use according to claim 6, characterized in that The genetically engineered Pichia pastoris is inoculated into a YPD liquid culture medium and cultured overnight to obtain a seed solution. The seed solution is then inoculated into a YPD liquid culture medium containing an immobilized carrier for immobilized fermentation. The supernatant fermentation solution is discarded, and lactose is used as a substrate to batch catalyze the production of oligomeric galactose from lactose.

8. The use according to claim 7, characterized in that The seed liquid was inoculated into the YPD liquid medium containing the immobilized carrier so that the initial OD of the Pichia pastoris genetically engineered bacteria was 600 It is 1.0~5.

0.

9. The use according to claim 7, characterized in that The specific conditions of the immobilized fermentation are: immobilized fermentation culture is carried out at 28-30°C and 220-250rpm for 90-150h; the batch catalysis of lactose to produce oligomeric galactose has a catalytic time of 12-24h for each batch and a catalytic temperature of 45-65°C.

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