Pichia pastoris genetically engineered strain for efficient surface display of enzyme based on adhesion protein, and construction method therefor and use thereof

Abstract

Disclosed in the present invention are a Pichia pastoris genetically engineered strain for the efficient surface display of an enzyme based on an adhesion protein, and a construction method therefor and the use thereof in immobilized continuous catalysis. An adhesion protein gene, a β-galactosidase gene LacA, and a cell wall anchoring protein gene Pir1p are expressed in the starting strain, Pichia pastoris. The Pichia pastoris genetically engineered strain is obtained by integrating and expressing a cell wall adhesion protein gene into the genome of Pichia pastoris GS115, integrating a β-galactosidase surface display gene in multiple copies, and displaying a target protein, β-galactosidase, on a yeast surface under the control of a constitutive promoter. The Pichia pastoris genetically engineered strain is capable of performing multi-batch continuous catalysis, and exhibits a catalytic effect superior to that of the enzyme surface display genetically engineered strain without an integrated biofilm gene.

Description

A Pichia pastoris genetically engineered strain for efficient surface display of adhesion proteins, its construction method, and its applications. Technical Field

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a Pichia pastoris genetically engineered strain with high-efficiency surface display of enzymes based on adhesion proteins, its construction method, and its application. Background Technology

[0002] Currently, green and sustainable biomanufacturing is the core of achieving energy conservation and emission reduction, waste value-added processing, and the reuse of renewable resources in large-scale industrial production. The development of cell immobilization technology is a major factor supporting cost reduction, efficiency improvement, and continuous operation in biomanufacturing processes. Biomembrane immobilization technology primarily relies on the spontaneous aggregation and adhesion of microbial cells, encapsulated by an extracellular matrix (polysaccharides, proteins, amyloid proteins, lipids, and extracellular DNA), to a carrier surface, forming a natural biological community. This natural microbial community exhibits high cell viability, cell regeneration capacity, and high tolerance to external stimuli. This immobilization strategy, with its enormous development potential, has been used in processes such as continuous fermentation of amino acids by *E. coli*, yeast fermentation of ethanol, and phytase production by *Pichia pastoris*. However, few studies have applied biomembrane immobilization strategies to continuous biocatalytic processes.

[0003] Traditional biocatalysis processes are severely limited by cumbersome enzyme purification processes, enzyme instability, and difficulty in recovery from the reaction medium. To address these drawbacks, whole-cell biocatalysis strategies involving enzyme display on microbial surfaces have been proposed. Displayed enzymes coexist in a relatively stable cellular environment, effectively resisting changes in the external environment and maintaining good catalytic activity. This whole-cell approach also eliminates expensive enzyme purification steps and facilitates enzyme recovery and reuse, thereby comprehensively improving the overall sustainability of biocatalysis applications. Furthermore, displaying enzymes directly expose them to the substrate, avoiding insufficient cellular mass transfer caused by transmembrane transport of large substrates and excessive intracellular transport. Nevertheless, the catalytic capacity of displayed enzymes largely depends on the surface enzyme density. Display efficiency can be improved through host modification, directed enzyme evolution, copy number optimization, and single-cell level enzyme saturation loading to achieve high-efficiency catalysis at higher enzyme densities. However, continuous catalysis studies utilizing the high cell density of biomembrane clusters to improve enzyme density at overall cell density have not yet been reported.

[0004] Pichia pastoris, as a superior host for heterologous proteins and industrial enzymes in industrial production, boasts advantages such as high cell culture density, high-intensity expression, post-translational protein processing, and absence of endotoxins and viral contamination. However, its biofilm formation ability is relatively weak, revealing problems such as unstable clustering and easy detachment of adsorbed material in actual fermentation processes. Previous work has explored some genes conducive to film formation using genetic engineering methods, making them suitable for continuous immobilized fermentation. This prompts those skilled in the art to further explore potential biofilm genes, investigate the performance of Pichia pastoris adhesion carrier media, and expand its application in immobilized continuous catalysis by combining it with surface-display whole-cell catalysis methods, thereby achieving the goal of 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 shortcomings of the prior art by providing a Pichia pastoris genetically engineered strain that efficiently displays β-galactosidase on the surface based on adhesion proteins. The Pichia pastoris genetically engineered strain can express adhesion protein genes, enhancing its ability to form biofilms. It also has an expression cassette that integrates multiple copies of the fusion gene of β-galactosidase gene LacA and cell wall anchoring protein gene Pir1p, thereby controlling the display of the target protein β-galactosidase on the surface of Pichia pastoris. This allows it to be applied to the immobilized continuous catalysis of lactose to galactooligosaccharide, achieving the goal of shortening batch catalysis time and recycling natural catalysts.

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

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

[0008] To address the first technical problem mentioned above, this invention discloses a Pichia pastoris genetically engineered strain that efficiently displays enzymes based on adhesion proteins. The strain heterologously expresses adhesion protein genes, β-galactosidase gene LacA, and cell wall anchoring protein gene Pir1p in the starting strain of Pichia pastoris.

[0009] The Pichiapastoris mentioned above is Pichiapastoris GS115.

[0010] The adhesion protein is a potential protein containing adhesion functional protein domains (Flo11p domain, cellulose-binding domain, GLEYA lectin-like binding domain, or lectin Flo9 type III domain) or a mutant lacking these adhesion functional domains; the adhesion protein gene is any one of flo1, flo2, flo3, flo4, flo5-2, flo11, gcw16, gcw16ΔFlo11p, and gcw16ΔCBD; the NCBI accession numbers for 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 Nos. 1 to 4; the genes gcw16ΔFlo11p and gcw16ΔCBD are truncated versions of the gene gcw16. Compared with the gcw16 encoded protein, the proteins encoded by gcw16ΔFlo11p and gcw16ΔCBD respectively lack the N-terminal 32-160 amino acids and 230-424 amino acids of the gcw16 encoded protein.

[0011] The nucleotide sequence of the β-galactosidase gene LacA is shown in SEQ No. 5; the nucleotide sequence of the cell wall anchoring 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 strain, wherein the expression cassette of the fusion gene Pir1p, which is a cell wall anchoring protein gene, 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] The expression cassette of the fusion gene Pir1p-LacA is integrated into the genome of the Pichia pastoris, either as a single copy or multiple copies; the copy number of the multiple copies is preferably 2 to 4.

[0014] The expression cassette of the fusion gene Pir1p-LacA is regulated by a constitutive promoter, preferably the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP).

[0015] Specifically, the method for constructing the Pichia pastoris genetically engineered strain is as follows:

[0016] (1) An expression cassette for the fusion gene Pir1p-LacA was constructed using overlap PCR (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, the expression cassette of the fusion gene Pir1p-LacA obtained in step (1) was integrated into the Pichia pastoris genome to obtain Pichia pastoris genetically engineered strains with β-galactosidase on the 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, the expression cassette of the adhesion gene was obtained 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 strain with surface-displaying β-galactosidase obtained in step (2) to obtain Pichia pastoris genetically engineered strain with surface-displaying β-galactosidase based on adhesion protein.

[0021] To address the second technical problem mentioned above, the present invention further discloses the application of the Pichia pastoris genetically engineered strain in the continuous immobilization of lactose to galactooligosaccharide production.

[0022] Specifically, the Pichia pastoris genetically engineered strain was inoculated into YPD liquid medium and cultured overnight to obtain a seed culture. The seed culture was then inoculated into YPD medium containing an immobilization carrier for immobilization fermentation. The supernatant fermentation broth was discarded, and batch catalytic lactose to galacto-oligosaccharide was carried out using lactose as a substrate.

[0023] In this process, the seed culture is inoculated into YPD liquid medium containing an immobilized carrier to induce an initial OD of the Pichia pastoris genetically engineered strain. 600 The value ranges from 1.0 to 5.0.

[0024] The immobilized fermentation is specifically carried out under the following conditions: immobilized fermentation culture at 28-30℃ and 220-250rpm for 90-150h; the batch catalytic lactose to galactooligosaccharide production process is carried out with a catalytic time of 12-24h per batch and a catalytic temperature of 45-65℃ per batch.

[0025] The immobilization carrier includes cotton fiber, polypropylene, polyethylene, loofah, polyester fiber, bacterial cellulose membrane, bamboo fiber, and central control fiber membrane, etc.

[0026] The YPD liquid culture medium consists of 10–15 g / L yeast extract, 15–25 g / L peptone, and 15–25 g / L glucose.

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

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

[0029] (1) The Pichia pastoris genetically engineered strain based on adhesion protein for efficient surface display of enzymes disclosed in this invention expresses the adhesion protein gene by integrating it into the Pichia pastoris genome. During static and dynamic culture, the Pichia pastoris genetically engineered strain has enhanced cell biofilm formation ability and stronger adsorption capacity in immobilized fermentation culture, thereby increasing 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 strain is increased by about 17.0%.

[0030] (2) The Pichia pastoris genetically engineered strain disclosed in this invention uses a constitutive promoter to regulate the expression of the fusion gene Pir1p-LacA, which is a cell wall anchoring protein gene Pir1p and a β-galactosidase gene LacA. This allows the exogenous fusion gene Pir1p-LacA to be expressed as the Pichia pastoris cells grow, thus displaying β-galactosidase on the surface of the Pichia pastoris cells. There is no need to add exogenous methanol, which has cytotoxicity, to induce the expression of the fusion gene Pir1p-LacA.

[0031] (3) The Pichia pastoris genetically engineered strain disclosed in this invention integrates multiple copies of the expression cassette of the fusion gene Pir1p-LacA, which is a cell wall anchoring protein gene and a β-galactosidase gene LacA, into the Pichia pastoris genome. The cellular enzyme activity of β-galactosidase gradually increases with the increase of the number of copies of the expression cassette. In the catalytic process with lactose as a substrate, the catalytic effect of the Pichia pastoris genetically engineered strain with 2 copies of the expression cassette has reached saturation.

[0032] (4) The Pichia pastoris genetically engineered strain disclosed in this invention has both film-forming adsorption capacity and enzyme high-efficiency display capacity. It can be continuously catalyzed by immobilization of lactose to galactooligosaccharide in multiple batches, and the catalytic effect can maintain a certain stability. After 6 batches of catalysis, the relative conversion rate of lactose is still 78%, which shows good industrialization prospects in continuous catalysis. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

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

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

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

[0037] Figure 4 shows the biofilm formation of the original Pichia pastoris GS115 and the Pichia pastoris genetically engineered strains (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 3, as well as the staining at the bottom of the well plates.

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

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

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

[0041] Figure 8 is a nucleic acid electrophoresis diagram of the enzyme display expression cassette in Example 7; in which 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; and lane 5 is the DNA marker of DL5000.

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

[0043] Figure 10 shows the cellular enzyme activity and single-batch yield of galactooligosaccharide produced from lactose in the genetically engineered bacteria after integration of the adhesion protein gene in Example 8. Figure a shows the cellular enzyme activity, and Figure b shows the yield of galactooligosaccharide.

[0044] Figure 11 shows the yield of the genetically engineered bacteria based on adhesion proteins in Example 9, which catalyzes the production of galactooligosaccharides from lactose in a continuous batch immobilization process.

[0045] Figure 12 shows the relative conversion rate of lactose conversion catalyzed by the high-efficiency enzyme display genetically engineered bacteria based on adhesion proteins in Example 9 during continuous immobilization batches. Detailed Implementation

[0046] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0047] The plasmids, restriction enzymes, high-fidelity enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations were performed according to the kit instructions. Other experimental consumables, unless otherwise specified, can be obtained commercially. The preparation of competent cells, colony PCR, nucleic acid agarose gel electrophoresis, electroporation, and extraction and preservation of yeast genome were performed according to standard methods.

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

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

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

[0051] The following examples illustrate the determination of lactose, galactooligosaccharide, glucose, and galacose concentrations in the catalyst solution: 1 mL of the catalyst solution was boiled at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was diluted a certain factor and filtered through a 0.22 μm filter membrane. High-performance liquid chromatography (HPLC) was used to analyze the sugar concentrations of each component in the catalyst system. HPLC detection method: differential refractive index detector; chromatographic column: BioRad HPX-87H (9 μm, 300 mm × 7.8 mm); column temperature: 60℃; mobile phase: 5 mmol / L H₂SO₄ aqueous solution; flow rate: 0.4 mL / min; injection volume: 5 μL.

[0052] The YPD liquid culture medium described in the following examples has a formulation of 10-15 g / L yeast extract, 15-25 g / L peptone, and 15-25 g / L glucose, and the YPD solid culture medium has a formulation of 10-15 g / L yeast extract, 15-25 g / L peptone, 15-25 g / L glucose, and 15-25 g / L agar.

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

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

[0055] (1) Amplification of the target gene fragments: Using the Pichia pastoris GS115 genome as a template, the gene fragments gcw16, flo1, flo2, flo3, flo4, flo5-2, and flo11 were amplified using primers for each target gene and then recovered by gel electrophoresis. The agarose gel electrophoresis images of the above gene fragments are shown in Figure 1a.

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

[0057] (3) Construction of linearized recombinant plasmid fragments: The adhesion gene fragment obtained in step (1) was ligated with the vector fragment obtained in step (2) in one-step cloning and transformed into E. coli DH5α competent cells. Then, the cells were plated on LB agar plates containing bleomycin. Single colonies were picked, plasmids were extracted, and sequenced. The plasmids with correct sequencing were the pGAPZαA plasmids with successfully inserted adhesion protein genes, and 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 digested with restriction endonuclease ArVII to form linearized recombinant plasmids, and the linearized recombinant plasmids were purified by gel extraction.

[0058] (4) Construction of Pichia pastoris genetically engineered strains: Using Pichia pastoris GS115 as the starting strain, the linearized plasmids obtained in step (3) were electroporated into Pichia pastoris GS115 competent cells, and then plated on YPD plates (containing 100mM bleomycin) and cultured at 30℃ and 250rpm until single colonies appeared. The single colonies grown on the plates were verified by colony PCR, and positive transformants were screened and their genes were sequenced. In this example, a total of 7 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] Example 2: Construction of a Pichia pastoris genetically engineered strain based on the gcw16 truncated gene

[0061] Figure 2 shows 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.

[0062] Recombinant expression plasmids of the truncated gcw16 genes 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 whole plasmid genome was integrated and expressed at the pGAP site of the promoter using a homologous recombination system. The primer sequences involved are shown in Table 2. The specific construction steps are as follows:

[0063] (1) Amplification of the target gene fragments: Using the Pichia pastoris GS115 genome as a template, the double fragments of the target gene gcw16ΔFlo11p were amplified using primers gcw16-F / gcw16ΔFlo11p-R and gcw16ΔFlo11p-F / gcw16-R, and the double fragments of the target gene gcw16ΔCBD were amplified using primers gcw16-F / gcw16ΔCBD-R and gcw16ΔCBD-F / gcw16-R, and then recovered by gel electrophoresis. The agarose gel electrophoresis images of the above target gene fragments are shown in Figure 3a.

[0064] (2) Amplification of plasmid fragments: Using the original pGAPZαA as a template and GAPαA-F / GAPαA-R as primers, PCR amplification was performed. The amplification product was digested with restriction endonuclease Dpn I to eliminate the excess circular pGAPαA plasmid and obtain the fragment of the vector pGAPZαA. Its agarose gel electrophoresis diagram is shown in Figure 3a.

[0065] (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) in one step and transformed into E. coli DH5α competent cells. Then, the cells were plated on LB agar plates containing bleomycin, and single colonies were picked to extract plasmids and sequenced. The plasmids with correct sequencing were the pGAPZαA plasmids with successfully inserted target genes, and 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 purified by gel extraction.

[0066] (4) Construction of Pichia pastoris genetically engineered strains: Using Pichia pastoris GS115 as the starting strain, the linearized plasmids obtained in step (3) were electroporated into Pichia pastoris GS115 competent cells, and then plated on YPD plates (100mM bleomycin) and cultured at 30℃ and 250rpm until single colonies appeared. The single colonies grown on the plates were verified by colony PCR, and positive transformants were screened and gene sequencing was performed. As shown in Figure 3b, compared with the original Pichia pastoris band (about 1100bp), 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.

[0067] Table 2 Primer sequences required for constructing recombinant plasmids of the gcw16 truncated gene

[0068] Example 3 Crystal Violet Staining Experiment

[0069] (1) The original Pichia pastoris GS115 and all Pichia pastoris genetically engineered strains with integrated adhesion genes in Examples 1 and 2 were inoculated into 5 mL of sterile YPD liquid medium and cultured overnight to activate them;

[0070] (2) Inoculate the activated bacterial solution from step (1) into 100 mL of YPD liquid medium at an inoculation rate of 1%, and continue to culture at 30 °C and 250 rpm until the bacterial solution reaches OD. 600 The value is between 0.8 and 1.2;

[0071] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterile YPD liquid culture medium to make the OD of the diluted bacterial solution... 600 It is 0.01;

[0072] (4) Take 200 μL of diluted bacterial solution and add it to a 96-well plate. Use YPD liquid medium as a control and incubate at 30℃ for 2, 3 and 5 days.

[0073] (5) Pour out the bacterial culture from the 96-well plate, wash the 96-well plate 2-3 times with 200 μL of 0.01M PBS buffer, and pat dry;

[0074] (6) Add 200 μL of 0.1% w / v crystal violet aqueous solution (1 g / L) to the 96-well plate in step (5), stain for 10-20 min, then rinse 2-3 times with 0.01 M PBS buffer and pat dry;

[0075] (7) Add 200 μL of glacial acetic acid to the 96-well plate from step (5) to dissolve the biofilm, gently shake for 40 min, and measure the OD.560 (OD 560 The value reflects the amount of biofilm formation, and the average value is taken.

[0076] Biofilm formation rate (OD) of Pichia pastoris genetically engineered strains integrating different adhesion protein genes 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 formation of Pichia pastoris biofilms. Among them, the adhesion protein gene gcw16 and its truncated genes gcw16ΔFlo11p and gcw16ΔCBD significantly improved its biofilm-forming ability. Figure 4 shows the biofilm formation statistics of the original Pichia pastoris (GS115) and the engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) cultured for 5 days in this example, as well as the staining diagram at the bottom of the well plate. It can be seen from the figure that the gcw16ΔFlo11p gene, after truncating the gcw16 gene, is more conducive to the formation of biofilm structures by Pichia pastoris on the well plate (polyethylene material).

[0077] Table 3. Biofilm formation rate of Pichia pastoris genetically engineered strains with integrated adhesion protein genes.

[0078] Example 4 Plate invasion and plate growth experiments

[0079] (1) The original Pichia pastoris GS115, the +gcw16 constructed in Example 1, the +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were respectively inoculated into 5 mL of sterile YPD liquid medium and cultured overnight to activate them;

[0080] (2) Inoculate the activated bacterial solution from step (1) into 100 mL of YPD liquid medium at a 1% inoculation rate, and continue culturing at 30°C and 250 rpm until the bacterial solution reaches OD. 600 The value is between 0.8 and 1.2;

[0081] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterile YPD liquid culture medium to make the OD of the diluted bacterial solution... 600 The values ​​are 1, 0.1, 0.01, and 0.001.

[0082] (4) Take 2 μL of the diluted bacterial solution and drop it vertically onto a YPD solid medium plate. After incubating at 30°C for 1 day, record the growth status of each concentration of the strain on the plate; at the same time, take OD values ​​of each strain. 600 10 μL of bacterial suspension with a concentration of 1 was dropped onto a YPD solid medium plate and incubated at 30°C for 5 days. The invasive ability of each strain on the plate was then recorded.

[0083] The experimental results are shown in Figure 5. Compared with the original Pichia pastoris GS115, the Pichia pastoris genetically engineered strain with integrated adhesion protein gene did not have enhanced plate invasion ability, and the integrated expression of the gene did not inhibit the growth of Pichia pastoris itself.

[0084] Example 5: Cell Counting Experiment Using Well Plate Adsorption under Static Culture

[0085] (1) The original Pichia pastoris GS115, the +gcw16 constructed in Example 1, the +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were respectively inoculated into 5 mL of sterile YPD liquid medium and cultured overnight to activate them;

[0086] (2) Inoculate the activated bacterial solution from step (1) into 100 mL of YPD liquid medium at a 1% inoculation rate, and continue culturing at 30°C and 250 rpm until the bacterial solution reaches OD. 600 The value is between 0.8 and 1.2;

[0087] (3) Take 2 mL of the bacterial solution obtained in step (2) and dilute it with sterile YPD liquid culture medium to make the OD of the diluted bacterial solution... 600 It is 0.01;

[0088] (4) Take 200 μL of the diluted bacterial solution and add it to a 96-well plate. Use YPD liquid medium as a control and incubate at 30°C for 3 days.

[0089] (5) Pour out the bacterial culture from the 96-well plate in a clean bench and wash with 200 μL of PBS buffer 2-3 times;

[0090] (6) Subsequently, the adherent cells in the 96-well plate were desorbed with 200 μL of 0.01 M sterile PBS buffer (containing 0.01 M proteinase K) for 30 min;

[0091] (7) Finally, the desorbed cell solution was serially diluted 100 times, 1000 times and 10000 times, and 100 μL of each solution was spread on YPD solid culture medium plates. After incubation at 30°C for 3 days, plate counts were performed. Three replicates were set up for each group and the average value was taken.

[0092] The experimental results are shown in Figure 6. Compared with the original Pichia pastoris GS115, the number of adherent cells in the genetically modified Pichia pastoris was significantly increased. Among them, the number of adherent cells in the genetically engineered Pichia pastoris overexpressing the truncated gene gcw16ΔFlo11p was 8.4 × 10⁻⁶. 4 The CFU / μL concentration is two orders of magnitude higher than that of the original Pichia pastoris GS115.

[0093] Example 6: Adsorption effect of Pichia pastoris genetically engineered bacteria during dynamic immobilization fermentation

[0094] (1) The original Pichia pastoris GS115, the +gcw16 constructed in Example 1, the +gcw16ΔFlo11p and +gcw16ΔCBD constructed in Example 2 were respectively inoculated into 5 mL of sterile YPD liquid medium and cultured overnight to activate them;

[0095] (2) Inoculate the activated bacterial solution from step (1) into 100 mL of YPD liquid medium at an inoculation rate of 1%, and continue to culture at 30°C and 250 rpm for 24 h.

[0096] (3) Then, the bacterial solution OD from step (2) 600 Adjusted to 1.0, the medium was 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 density of free cells in the fermentation supernatant was recorded every 12 h. Meanwhile, on days 1, 2, and 3, the cotton fiber medium adsorbed with microbial cells was removed, 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.

[0097] The experimental results are shown in Figure 7. In static culture, the +gcw16ΔFlo11p strain, which exhibited the best film-forming effect, showed a similar trend in the free cell content of its dynamic fermentation supernatant to the original Pichia pastoris GS115, both showing a high number of planktonic cells. In contrast, the fermentation supernatants of the +gcw16 and +gcw16ΔCBD strains did not contain excessive free cells, and the dry weight of the microbial cells adsorbed onto the cotton fiber medium was significantly higher than that of the original Pichia pastoris GS115 and +gcw16ΔFlo11p strains. After 3 days of culture, compared with the original Pichia pastoris GS115, the number of cells adsorbed on the carrier by +gcw16 and +gcw16ΔCBD increased by approximately 17.0%. These results indicate that the Pichia pastoris genetically engineered strain based on adhesion proteins constructed in this invention can also be applied to dynamic immobilized fermentation culture, and the immobilized fermentation adsorption effect is more robust and less prone to detachment.

[0098] Example 7: Construction of a Pichia pastoris genetically engineered strain displaying β-galactosidase on its surface

[0099] An expression cassette, Pir1p-LacA (derived from patent CN114606151A), was constructed using the constitutive promoter pGAP of Pichia pastoris. This fusion gene, representing the anchoring protein gene Pir1p and the β-galactosidase gene LacA, was then integrated into the Pichia pastoris genome using homologous recombination technology and a gene editing system (a CRISPR / Cas9 system was used in this embodiment to address the need for multiple rounds of genome editing). This resulted in a fermentation system where enzyme display and expression were coupled to cell growth, effectively avoiding the cytotoxicity and environmental pollution associated with the inducer methanol. The primers involved are shown in Table 4, and the specific construction steps are as follows:

[0100] (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 obtained by amplification using overlap PCR with primers pGAP-F / AOXTT-R, and then purified by gel extraction.

[0101] (2) Using the Pichia pastoris GS115 genome as a template, the upper and lower homologous arms I-2-Up and I-2-Down of the site (PNSI-2) were amplified using primers PNSI-2-UF / PNSI-2-UR and PNSI-2-DF / PNSI-2-DR. Finally, the upper and lower homologous arm fragments were overlapped with the gene expression cassette constructed in step (1) by primers PNSI-2-UF / PNSI-2-DR to obtain the Donor DNA fragment, which was then purified by gel extraction.

[0102] (3) 5 μg of the Donor DNA fragment purified in step (2) and 1 μg of CRISPR / Cas9 plasmid containing the PNSI-2 site were electroporated into Pichia pastoris competent cells. The cells were then plated on YPD solid medium 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 to verify their growth. Positive transformants were screened and their genes were sequenced. Multiple passages were performed to remove the tool plasmid. Finally, the Pichia pastoris genetically engineered strain +1*Pir1p-LacA was successfully constructed.

[0103] (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 genetically engineered strains +2*Pir1p-LacA, +3*Pir1p-LacA, and +4*Pir1p-LacA were successfully constructed.

[0104] The nucleotide sequences of the Pichia pastoris genomic loci PNSI-2, PNSⅠ-3, PNSⅡ-4 and PNSⅢ-5 are shown in SEQ No. 7 to 10, respectively.

[0105] Figure 8 is a nucleic acid electrophoresis diagram of the enzyme display expression cassette in this embodiment.

[0106] The constructed multi-copy enzyme-displaying genetically engineered bacteria were fermented and cultured, and their enzyme activity was detected. The specific fermentation method was as follows: single cloning sites of the above-described Pichia pastoris genetically engineered strains (+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 size, the culture was transferred to 50 mL of YPD liquid medium and cultured further at 30℃ and 250 rpm; the enzyme activity in the fermentation broth was detected every 24 hours.

[0107] Cellular enzyme activity was detected using the method described in patent CN114606151A, as follows: o-nitrophenyl-β-D-galactoside (oNPG) was used as a substrate, and an appropriate amount of enzyme-displaying genetically engineered bacterial cells were added. 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.

[0108] The cell enzyme activity detection results are shown in Figure 9. The multi-copy enzyme display genetically engineered strain constructed above can display β-galactosidase secretion on the cell wall without the addition of an inducer, as the yeast cells grow; moreover, the activity of the display enzyme gradually increases with the increase of the copy number of the enzyme display gene expression cassette.

[0109] Table 4 Primer sequences required for constructing Pichia pastoris genetically engineered enzymes displaying β-galactosidase on their surface.

[0110] Example 8: Construction of Pichia pastoris genetically engineered strain with high-efficiency surface display of β-galactosidase based on adhesion proteins and verification of its single-batch catalytic effect.

[0111] The construction of Pichia pastoris genetically engineered strains using β-galactosidase based on adhesion proteins for efficient surface display is as follows:

[0112] (1) Using the recombinant plasmid pGAPZαA-gcw16ΔCBD from Example 2 as a template, the corresponding expression cassette pGAP-gcw16ΔCBD-AOXTT was amplified using primers pGAP-F / pGAP-R and purified by gel extraction; then, using the Pichia pastoris GS115 genome as a template, the upper and lower homologous arms IV-2-Up and IV-2-Down of this 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 homologous arm fragments were overlapped with the gene expression cassette by primers PNSⅣ-2-UF / PNSⅣ-2-DR to obtain the Donor DNA fragment, which was then purified by gel extraction.

[0113] (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 competent cells of the multicopy enzyme-displaying Pichia pastoris genetically engineered strain in Example 7. The cells were then plated on 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 to verify their growth, and positive transformants were screened and their genes were sequenced. Finally, the following Pichia pastoris genetically engineered strains based on adhesion protein-dependent β-galactosidase, +gcw16ΔCBD,1*Pir1p-LacA, +gcw16ΔCBD,2*Pir1p-LacA, +gcw16ΔCBD,3*Pir1p-LacA, and +gcw16ΔCBD,4*Pir1p-LacA, were successfully constructed.

[0114] The nucleotide sequence of the Pichia pastoris genomic locus PNSⅣ-2 is shown in SEQ No. 11.

[0115] The primer sequences required for the above construction process are shown in Table 5.

[0116] The recombinant bacteria displaying the multicopy enzyme based on the membrane-forming gene constructed above were fermented and cultured, and their enzyme activity was detected. The specific fermentation culture method was as follows: the single cloning site of the above-mentioned genetically engineered bacteria was transferred to 5 mL of YPD liquid medium and cultured overnight for activation; then, at an inoculum volume of 1%, it was transferred to 50 mL of YPD liquid medium and cultured for a further period of time at 30℃ and 250 rpm. The cellular enzyme activity in the fermentation broth was recorded after 120 h.

[0117] The recombinant bacteria displaying the multicopy enzyme based on the membrane-forming gene constructed above were fermented and cultured, and then subjected to single-batch catalysis using lactose as a substrate. The catalytic conditions were 400 g / L lactose, 50 °C, and a catalytic reaction time of 24 h. The content of galactooligosaccharides in the catalytic solution was detected by HPLC.

[0118] The results of cellular enzyme activity and polygalactosyl (GOS) production are shown in Figure 10. Figure 10a shows that after integrating the truncated gene gcw16ΔCBD expressing the adhesion protein into recombinant strains with different copy numbers of the enzyme display gene, the cellular enzyme activity of β-galactosidase was slightly increased compared to recombinant strains without gcw16ΔCBD integration. This may be because the GPI binding site of the adhesion protein encoded by gcw16ΔCBD targets the cell wall, causing changes in the cell wall structure of the recombinant strains and resulting in an increase in the anchored enzyme protein. Figure 10b shows that in a single-batch catalysis process using lactose as a substrate, after integrating gcw16ΔCBD into a genetically engineered strain with a single copy of the integrase display gene, the production of galacto-oligosyl (GOS) significantly increased by 17.8% compared to recombinant strains without gcw16ΔCBD integration. However, the expected increase in GOS production did not occur as the copy number of the enzyme display gene increased. When the copy number of the enzyme display gene was 2, the catalytic efficiency of the recombinant strain in producing GOS from lactose reached saturation.

[0119] Table 5 Primer sequences required for constructing genetically engineered strains in Example 8.

[0120] Example 9: Application of Pichia pastoris genetically engineered strains using adhesion protein-based β-galactosidase for efficient surface display in immobilized continuous batch catalytic production of galactooligosaccharides from lactose.

[0121] To investigate the catalytic efficiency of the enzyme-displaying genetically engineered bacteria based on adhesion proteins in immobilized continuous batch catalysis, this example used the Pichia pastoris genetically engineered strain +gcw16ΔCBD,2*Pir1p-LacA, which 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 immobilization fermentation culture steps in Example 6 to allow the strain to be fully adsorbed on the cotton fiber carrier. Then, this immobilized cell medium was placed in the 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 sugar content in the catalytic system and catalytic solution was detected by the same method as in Example 8.

[0122] In the formula, M(GOS3) and M(GOS4) refer to the concentrations (g / L) of galactooligosaccharides with degrees of polymerization of 3 and 4 at a certain time during the catalytic reaction; M(Lac0) refers to the initial concentration (g / L) of lactose added to the catalytic system; GOS conversion rate (+gcw16ΔCBD,2*Pir1p-LacA,batch 1) refers to the GOS conversion rate of the first batch in a series of batches catalyzed with strain +gcw16ΔCBD,2*Pir1p-LacA to produce galactooligosaccharides from lactose. GOS conversion rate (batch n) refers to the GOS conversion rate of the nth batch in a series of batches catalyzed with strain +gcw16ΔCBD,2*Pir1p-LacA or +gcw16ΔCBD to produce galactooligosaccharides from lactose.

[0123] Figure 11 shows the yield results of six consecutive batches of catalytic lactose to galactooligosaccharide (GOS) production. Unlike Example 8, the yield of GOS from the lactose-catalyzed strain +gcw16ΔCBD,2*Pir1p-LacA (a 2-copy enzyme display strain integrating the gcw16ΔCBD gene) was significantly higher than that from the strain +2*Pir1p-LacA (a 2-copy enzyme display strain without the gcw16ΔCBD gene integration). This indicates that integrating the gcw16ΔCBD gene facilitates cell adhesion to the cotton fiber carrier, increasing enzyme density and thus enhancing the catalytic effect. Figure 12 shows the relative conversion rate of lactose conversion catalyzed by +gcw16ΔCBD,2*Pir1p-LacA in consecutive immobilized batches. In terms of relative conversion rate, after six batches of catalysis, the relative conversion rate of the 2-copy enzyme display strain integrating the gcw16ΔCBD gene remained at approximately 76%.

[0124] This invention provides a Pichia pastoris genetically engineered strain based on adhesion protein-displayed enzyme surface, along with its construction concept and method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A Pichia pastoris genetically engineered strain for efficient surface display of enzymes based on adhesion proteins, characterized in that, In the starting strain *Pichia pastoris*, an adhesion protein gene is homologously expressed, a β-galactosidase gene *LacA* is heterologously expressed, and a cell wall anchoring protein gene *Pir1p* is heterologously expressed. 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 anchoring protein gene *Pir1p* is shown in SEQ No.

6. *gcw16ΔCBD* is a truncated version of *gcw16*, and the protein encoded by *gcw16ΔCBD* lacks the N-terminal 230–424 amino acids of the protein encoded by *gcw16* compared to the protein encoded by *gcw16*.

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

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

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

5. The construction method according to claim 3, characterized in that, The expression cassettes of the fusion gene Pir1p-LacA and the adhesion protein gene are both constitutively regulated by promoters.

6. The application of the Pichia pastoris genetically engineered strain according to claim 1 or 2 in the continuous immobilization of lactose to galactooligosaccharide production.

7. The application according to claim 6, characterized in that, The Pichia pastoris genetically engineered strain was inoculated into YPD liquid medium and cultured overnight to obtain a seed culture. The seed culture was then inoculated into YPD liquid medium containing an immobilization carrier for immobilization fermentation. The supernatant fermentation broth was discarded, and batch catalytic lactose to galactooligosaccharide was carried out using lactose as a substrate.

8. The application according to claim 7, characterized in that, The seed culture was inoculated into YPD liquid medium containing an immobilized carrier to induce an initial OD of the Pichia pastoris genetically engineered strain. 600 The value ranges from 1.0 to 5.

0.

9. The application according to claim 7, characterized in that, The immobilized fermentation is carried out under the following conditions: immobilized fermentation culture at 28-30℃ and 220-250rpm for 90-150h; the batch catalytic lactose to galactooligosaccharide production is carried out with a catalytic time of 12-24h and a catalytic temperature of 45-65℃ per batch.

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