Pichia pastoris genetically engineered bacterium for enzyme efficient surface display based on adhesion protein as well as construction method and application of pichia pastoris genetically engineered bacterium

Abstract

The invention discloses pichia pastoris genetically engineered bacteria based on enzyme efficient surface display of adhesion protein, a construction method of the pichia pastoris genetically engineered bacteria and application of the pichia pastoris genetically engineered bacteria in immobilized continuous catalysis. An adhesion protein gene, a beta-galactosidase gene LacA and a cell wall anchoring protein gene Pir1p are heterologously expressed in starting strain pichia pastoris. According to the pichia pastoris genetically engineered bacterium, cell wall adhesion protein genes are integrated and expressed in a pichia pastoris GS115 genome, and the cell film-forming capacity of pichia pastoris is remarkably enhanced; and performing multi-copy integration on the beta-galactosidase surface display gene, and controlling the target protein beta-galactosidase to be displayed on the surface of the yeast by using a constitutive promoter. The pichia pastoris genetically engineered bacterium based on the high-efficiency surface display of the enzyme of the adhesion protein can be used for multi-batch continuous catalysis, the effect is better than that of the enzyme surface display genetically engineered bacterium which is not integrated with a biological membrane gene, the relative conversion rate is still kept at 78% after six batches, and the pichia pastoris genetically engineered bacterium has a good industrial application prospect in immobilized continuous catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a Pichia pastoris genetic engineering bacterium with high-efficiency surface display of enzyme based on adhesion protein, and a construction method and 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 the spontaneous aggregation and adhesion of microbial cells wrapped in extracellular matrix (polysaccharides, proteins, amyloid proteins, lipids and extracellular DNA) on 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 prepare 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 greatly limited by the cumbersome enzyme purification process, enzyme instability, and difficulty in recovering from the reaction medium. To address these drawbacks, whole-cell biocatalytic strategies for enzyme display on the surface of microorganisms have been proposed. The displayed enzyme coexists in a relatively stable cellular environment, effectively resisting changes in the external environment, and can maintain good enzyme catalytic activity. This whole-cell approach also solves the expensive enzyme purification steps and facilitates enzyme recycling and reuse, thereby comprehensively improving the overall sustainability of biocatalytic applications. In addition, the displayed enzyme is directly exposed to the substrate, avoiding the lack of cell mass transfer caused by the transmembrane transport of large substrates and excessive transport of intracellular substances. Nevertheless, the catalytic ability of the displayed enzyme depends largely on the surface enzyme density. The display efficiency can be improved by host modification, enzyme directed evolution, copy number optimization, and enzyme saturation loading at the single cell level to meet the requirements of high display enzyme density for efficient catalysis. However, in terms of overall cell density, continuous catalysis studies that use the characteristics of high cell density of biofilm clusters to improve enzyme density have not been reported.

[0004] As a superior host for heterologous proteins and industrial enzymes in industrial production, Pichia pastoris has the advantages of high cell culture density, high-intensity expression, post-translationally modified protein processing, and no endotoxin and virus contamination. However, its biofilm formation ability is relatively weak, and the actual fermentation process exposes problems such as unstable clusters and easy adsorption and shedding. In the previous work, genetic engineering methods have been used to explore some genes that are conducive to film formation and make them suitable for continuous fixed fermentation. This has prompted technicians in this field to comprehensively explore potential biofilm genes, explore the performance of Pichia pastoris adhesion carrier media, and expand its application in immobilized continuous catalysis by combining surface-displayed whole-cell catalysis methods, so as to achieve the purpose 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 provide a Pichia pastoris genetically engineered bacterium with high-efficiency surface display of β-galactosidase based on adhesion protein, wherein the Pichia pastoris genetically engineered bacterium can express adhesion protein genes to enhance the ability of its cells to form biofilms, and has multiple copies of an expression cassette of a fusion gene of the β-galactosidase gene LacA and the cell wall anchor protein gene Pir1p integrated to control the display of the target protein β-galactosidase on the surface of the Pichia pastoris, thereby applying the Pichia pastoris to immobilized and continuous catalysis of lactose to produce oligosaccharides, thereby achieving the purpose 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 bacteria.

[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 catalysis of lactose to produce galacto-oligosaccharides.

[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 missing 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, and integrates 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 into the genome of the Pichia pastoris by 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 2-4.

[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 overlap PCR technology, construct an expression cassette of the fusion gene Pir1p-LacA (using the recombinant plasmid pPIC9k-pir1p-lacA in patent CN114606151A as a template, and obtaining the fusion gene Pir1p-LacA fragment 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 catalysis of lactose to produce galacto-oligosaccharides.

[0022] Specifically, the genetically engineered Pichia pastoris is inoculated into a YPD liquid culture medium, cultured overnight to obtain a seed solution, and then the seed solution is 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 lactose to produce oligosaccharides.

[0023] The seed solution is inoculated into a YPD liquid medium containing an immobilized carrier so that the initial OD 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 oligosaccharides has a catalytic time of 12-24h for each batch and a catalytic temperature of 45-65°C.

[0025] Among them, the immobilized 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 contains 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 itself, difficulty in adsorbing carriers, poor continuous effect, etc., thereby realizing the application of the genetically engineered Pichia pastoris in biofilm immobilization 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, stronger adsorption ability 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 disclosed in the present invention uses a constitutive promoter to regulate the expression of the fusion gene Pir1p-LacA of the cell wall anchor protein gene Pir1p and the β-galactosidase gene LacA, so that the exogenous fusion gene Pir1p-LacA can be expressed along with the growth of Pichia pastoris cells, and the β-galactosidase is displayed on the surface of Pichia pastoris cells, without the need for exogenous addition of 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 of the 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 with lactose as the substrate, the catalytic effect of the genetically engineered Pichia pastoris with 2 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 oligosaccharides, 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 in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0034] Figure 1 The agarose gel electrophoresis diagram of the recombinant plasmid and Pichia pastoris transformant 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 DNA marker of DL5000.

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

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

[0037] Figure 4 It is a statistical graph of the biofilm formation amount of the original Pichia pastoris GS115 and the genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) in Example 3 and a staining graph of the bottom of the well plate.

[0038] Figure 5 The figures are the plate invasion and plate growth results of the original Pichia pastoris GS115 and the 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 free cell OD600 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 It 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] Fig. 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 in Example 7 during 120 h of fermentation.

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

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

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

[0046] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0047] The plasmids, endonucleases, high-fidelity enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out according to the kit instructions. Other experimental consumables, unless otherwise specified, can be obtained from commercial sources. The following experimental operations such as preparation of competent cells, colony PCR, nucleic acid agarose gel electrophoresis, electrotransformation and extraction and preservation of yeast genomes are carried out according to conventional methods.

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

[0049] The genome-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] The method for determining the concentration of lactose, galacto-oligosaccharide, glucose and galactose in the catalytic solution in the following examples is as follows: 1 mL of the catalytic solution is boiled at 100°C for 10 min, centrifuged at 12000 r / min for 5 min, the supernatant is diluted a certain multiple, filtered through a 0.22 μm filter membrane, and the sugar concentration of each component in the catalytic system is analyzed using high performance liquid chromatography (HPLC). HPLC detection method: differential refractive index detector; chromatographic column is BioRad HPX-87H (9 μm, 300 mm×7.8 mm), column temperature is 60°C; mobile phase is 5 mmol / L H2SO4 aqueous solution, flow rate is 0.4 mL / min; injection volume is 5 μL.

[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 genetically engineered Pichia pastoris strains based on adhesion proteins

[0054] A plurality of recombinant expression plasmids carrying adhesion protein genes flo1, flo2, flo3, flo4, flo5-2, flo11, and gcw16 were constructed respectively; then, Pichia pastoris GS115 was used as the starting strain, and the whole plasmid genome was integrated and expressed at the promoter pGAP site using the homologous recombination system. 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 1 As shown in a.

[0056] (2) Amplification of vector fragment: 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 fragments of recombinant plasmids: The adhesion gene fragments obtained in step (1) were cloned and connected with the vector fragments obtained in step (2) and transformed into competent E. coli DH5α cells, and then spread on LB plates containing bleomycin, single colonies were picked to extract plasmids and sequenced, and the correct sequencing was the pGAPZαA plasmid with the adhesion protein gene successfully inserted, 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 cut into linearized recombinant plasmids with restriction endonuclease ArVII, and the linearized recombinant plasmids were recovered and purified by gel recovery.

[0058] (4) Construction of genetically engineered strains of Pichia pastoris: Using Pichia pastoris GS115 as the starting strain, the linearized plasmids obtained in step (3) were electrotransformed into competent cells of Pichia pastoris GS115, and then spread on a YPD plate (containing 100 mM bleomycin), cultured at 30° C. and 250 rpm until a single colony appeared, and colony PCR was performed on the single colony grown on the plate to verify the positive transformants and perform gene sequencing. In this embodiment, a total of 7 genetically engineered strains of Pichia pastoris 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 Pichia pastoris genetically engineered strain based on 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 of the truncated gene gcw16ΔFlo11p (the protein encoded by this gene lacks the Flo11p binding domain compared with the protein encoded by gcw16) and gcw16ΔCBD (the protein encoded by this gene lacks the CBD binding domain compared with the protein encoded by gcw16) of gcw16 were constructed respectively; then, using Pichia pastoris GS115 as the starting strain, the homologous recombination system was used to perform full plasmid genome integration expression at the promoter pGAP site. 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 genome of Pichia pastoris GS115 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 diagram is shown in Figure 2. Figure 3 As shown in a.

[0066] (3) Construction of linearized fragments of recombinant plasmids: The gcw16ΔFlo11p and gcw16ΔCBD gene fragments obtained in step (1) were cloned and connected with the vector fragment obtained in step (2) and transformed into Escherichia coli DH5α competent cells, and then spread on LB plates containing bleomycin, single colonies were picked to extract plasmids and sequenced, and the correct sequencing was the pGAPZαA plasmid with the target gene successfully inserted, which were named pGAPZαA-gcw16ΔFlo11p and pGAPZαA-gcw16ΔCBD, respectively. The above recombinant plasmids were cut into linearized recombinant plasmids with restriction endonuclease ArVII, 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 spread on YPD plates (100 mM bleomycin) and cultured at 30°C and 250 rpm until single colonies appeared. Single colonies grown on the plates were verified by colony PCR, positive transformants were screened, and gene sequencing was performed. 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 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 culture 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 reached 0. 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) Take 200 μL of the diluted bacterial solution and add it to a 96-well plate, use YPD liquid medium as a control, and culture at 30°C for 2 days, 3 days, 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 to 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 in step (5), stain for 10 to 20 min, rinse 2 to 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 min and measure the OD 560 (OD 560 The value reflects the amount of biofilm formation), and the average value was taken.

[0078] Biofilm formation (OD) of Pichia pastoris genetically engineered bacteria with 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 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 statistical graph of biofilm formation of the original Pichia pastoris (GS115) and genetically engineered Pichia pastoris (+gcw16, +gcw16ΔFlo11p, +gcw16ΔCBD) cultured for 5 days in this example and the staining graph of 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 a biofilm structure by Pichia pastoris on the well plate (polyethylene material).

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

[0080]

[0081] Example 4 Plate invasion and 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 culture 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 reached 0. 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 a 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 and cultured at 30°C for 5 days, and 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 culture 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 reached 0. 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) Take 200 μL of the diluted bacterial solution and add it to a 96-well plate, use YPD liquid medium as a control, and culture 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 to 3 times with 200 μL 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 culturing 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 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, which is 2 orders of magnitude higher than that of the original Pichia pastoris GS115.

[0096] Example 6 Adsorption effect of genetically engineered Pichia pastoris 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 culture 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 amount, and the culture was continued at 30° C. and 250 rpm for 24 h;

[0099] (3) Then, the OD value of the bacterial solution in step (2) was 600 The pH value was adjusted to 1.0, and the medium was transferred to 50 mL of YPD liquid medium containing 2 g of cotton fiber carrier for immobilized fermentation culture. The medium was dynamically cultured at 30 °C and 250 rpm, and 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 days, 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 trend of the free cell content change in the dynamic fermentation supernatant of the +gcw16ΔFlo11p strain with the best film-forming effect in static culture is consistent with that of the original Pichia GS115, and both show more planktonic cells; while the fermentation supernatant of the +gcw16 and +gcw16ΔCBD strains does not have too many free cells, and the dry weight of microbial cells adsorbed on the cotton fiber medium is also significantly higher than that of the original Pichia GS115 and +gcw16ΔFlo11p strains. After 3 days of cultivation, compared with the original Pichia GS115, the amount of cells adhered to the carrier of +gcw16 and +gcw16ΔCBD increased by about 17.0%. These results show that the Pichia genetic engineering bacteria based on adhesion protein constructed by the present invention can also be used for dynamic immobilized fermentation culture, and the immobilized fermentation adsorption effect is more solid and not easy to fall off.

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

[0102] The constitutive promoter pGAP of Pichia pastoris was used to construct the expression cassette of the fusion gene Pir1p-LacA (derived from patent CN114606151A) of the anchor protein gene Pir1p and the β-galactosidase gene LacA, and the expression cassette was integrated into the Pichia pastoris genome using homologous recombination technology and gene editing system (the gene editing system designed in this embodiment is CRISPR / Cas9 system because of the need for multiple rounds of genome editing), and a fermentation system of enzyme display expression coupled with cell growth was constructed, which effectively avoided 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.

[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 overlap PCR 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 competent cells, and then spread on YPD solid culture medium 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 genetic engineering 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 display 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 genome 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 display genetically engineered bacteria constructed above were fermented and cultured, and the enzyme activity of the enzyme display genetically engineered bacteria was detected. The specific fermentation culture method is: the single clone points of the above Pichia genetically engineered strain +1*Pir1p-LacA, +2*Pir1p-LacA, +3*Pir1p-LacA, +4*Pir1p-LacA were transferred to 5mL YPD liquid culture medium for overnight culture and activation; then transferred to 50mL YPD liquid culture medium according to 1% inoculation amount, and continued to be cultured at 30°C and 250rpm; the cell enzyme activity in the fermentation broth was detected every 24h.

[0110] The cell enzyme activity detection refers to the method in patent CN114606151A, which is as follows: using o-nitrophenyl-β-D-galactoside (oNPG) as a substrate, adding an appropriate amount of enzyme display genetically engineered bacterial cells, reacting at a catalytic temperature of 50°C for 10 minutes, and the enzyme activity unit 1U is defined as the amount of cells required to convert 1nmol of oNPG per minute.

[0111] The results of cell enzyme activity test were as follows Fig. 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 an inducer; moreover, as the number of copies 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 protein and verification of its single-batch catalytic effect

[0116] The construction of Pichia pastoris genetic engineering bacteria with high-efficiency surface display of β-galactosidase based on adhesion protein 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 by gel extraction; then, using the genome of Pichia pastoris GS115 as a template, the upper and lower homologous 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 homologous 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 recovered and purified by gel extraction.

[0118] (2) 5 μg of the purified Donor DNA fragment in 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 with high efficiency of surface display of β-galactosidase 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 display recombinant bacteria based on the film-forming gene constructed above were fermented and cultured, and the enzyme activity was tested. The specific fermentation culture method is: the single clone point of the genetically engineered bacteria above was transferred to 5mL YPD liquid culture medium for overnight culture and activation; then, according to the 1% inoculation amount, it was transferred to 50mL YPD liquid culture medium, and continued to be cultured at 30°C and 250rpm, and the cell enzyme activity in the fermentation broth was recorded at 120h.

[0122] After fermentation culture of the multi-copy enzyme display recombinant bacteria based on the film-forming gene constructed above, single batch catalysis was carried out with lactose as substrate, and the catalytic conditions were 400 g / L lactose, 50° C., and catalytic reaction for 24 hours. The content of oligogalactose in the catalytic solution was detected by HPLC.

[0123] The results of cell enzyme activity and polygalactose production were as follows Fig.10 As shown. Fig.10 As can be seen in a, after the integration of the truncated gene gcw16ΔCBD expressing the adhesion protein into the recombinant strains with different copy numbers of the enzyme display gene, the cellular enzyme activity of β-galactosidase was slightly increased 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. Fig.10 As can be seen in Figure b, in the single batch catalysis process with lactose as substrate, after integrating gcw16ΔCBD on the basis of the genetic engineering strain with a single copy of the integrated enzyme display gene, the production of galacto-oligosaccharides (GOS) increased significantly by 17.8% compared with the recombinant strain without gcw16ΔCBD integration. However, with the increase in the number of copies of the enzyme display gene, the expected increase in GOS production did not occur. When the number of copies of the enzyme display gene was 2, the catalytic efficiency of the recombinant strain to produce GOS from lactose had 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 protein in immobilized continuous batch catalysis of lactose to produce galacto-oligosaccharides

[0127] In order to investigate the catalytic efficiency of the enzyme display genetically engineered bacteria based on adhesion proteins in immobilized continuous batch catalysis, this example uses the Pichia pastoris genetically engineered bacteria +gcw16ΔCBD, 2*Pir1p-LacA that integrates 2 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 are cultured for 120 hours according to the immobilized fermentation culture steps in Example 6, so that the strain is fully adsorbed on the cotton fiber carrier, and then the immobilized cell medium is 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 is 24 hours, and the catalytic solution is replaced every 24 hours. The method for detecting the content of sugar components in the catalytic system and the catalytic solution is the same as in Example 8.

[0128]

[0129] Wherein, 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 the first batch in the continuous batches of lactose products catalyzed by the strain +gcw16ΔCBD, 2*Pir1p-LacA. GOS conversion rate (batch n) refers to the GOS conversion rate of the nth batch in the continuous batches of lactose products 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 Fig.11 As shown, unlike Example 8, the yield of lactose catalysis product galacto-oligosaccharide (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. Fig.12 is 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 integrating the gcw16ΔCBD gene remained at around 76%.

[0131] The present invention provides a Pichia pastoris genetic engineering bacterium based on adhesion protein enzyme surface display and its construction ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A genetically engineered bacterium of Pichia pastoris with efficient surface display of enzymes based on adhesion proteins, characterized in that: Adhesion protein genes, β-galactosidase gene LacA and cell wall anchor protein gene Pir1p were heterologously expressed in the starting strain Pichia pastoris.

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

3. The genetically engineered Pichia pastoris according to claim 1, characterized in that 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 shown in SEQ No.1 to 4, respectively; the nucleotide sequence of the β-galactosidase gene LacA is shown in SEQ No.5; and the nucleotide sequence of the cell wall anchor protein gene Pir1p is shown in SEQ No.

6.

4. The method for constructing the genetically engineered Pichia pastoris according to claims 1 to 3, characterized in that: The expression cassette of the fusion gene Pir1p-LacA of the cell wall anchor protein gene Pir1p and the beta-galactosidase gene LacA and the expression cassette of the adhesion protein gene are integrated into the genome of the Pichia pastoris by using homologous recombination technology.

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

6. The construction method according to claim 4, 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.

7. Use of the genetically engineered Pichia pastoris according to any one of claims 1 to 3 in the continuous immobilized catalytic production of galacto-oligosaccharides from lactose.

8. The use according to claim 7, characterized in that: The genetically engineered Pichia pastoris is inoculated into a YPD liquid culture medium, cultured overnight to obtain a seed solution, and then the seed solution is 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 lactose to produce oligosaccharides.

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

0.

10. The use according to claim 8, 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 oligosaccharides has a catalytic time of 12-24h for each batch and a catalytic temperature of 45-65°C.

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