Saccharase-based pichia pastoris antibiotic-free screening system and application thereof

Abstract

This invention discloses an antibiotic-free screening system for Pichia pastoris based on sucrose hydrolase and its application, belonging to the field of bioengineering technology. This invention utilizes a sucrose hydrolase derived from Aspergillus flavus. SucF As a novel bifunctional screening marker for Pichia pastoris, it can completely replace traditional antibiotic selection markers and endow recombinant Pichia pastoris strains with the ability to directly and efficiently utilize inexpensive sucrose. Further optimization yielded a high-performance triple mutant (T65A / R322Q / S323G) with a sucrose hydrolysis conversion rate significantly increased by 57% compared to the wild type. Simultaneously, combined with… SucF Sucrose successfully achieved efficient secretory expression of human lactoferrin and human osteopontin using a histidine auxotrophic dual-labeling system, which involved transcription, folding and transport of yeast endogenous helper proteins. No antibiotics were added throughout the process.

Description

Technical Field

[0001] This invention relates to an antibiotic-free screening system for Pichia pastoris based on sucrose hydrolase and its application, belonging to the field of bioengineering technology. Background Technology

[0002] Pichia pastoris ( Komagataella phaffii Pichia pastoris is one of the most widely used eukaryotic expression systems in industry and academia. It possesses core technological advantages such as high-density fermentation with large biomass, strong exogenous protein secretion capacity, and post-translational modifications similar to those in higher eukaryotes (e.g., glycosylation, disulfide bond formation), making it a crucial foundational cell for the manufacture of industrial enzymes, pharmaceutical proteins, and novel food additives. However, existing Pichia pastoris fermentation platforms face two serious bottlenecks in their evolution towards green, low-cost, and large-scale industrialization: First, an extreme reliance on antibiotic resistance marker selection systems. The screening and maintenance of conventional recombinant strains mainly rely on antibiotic resistance genes such as bleomycin, G418, or hygromycin. The use of antibiotics significantly increases the raw material costs of industrial fermentation and easily leads to antibiotic gene residues. This makes it impossible for related recombinant protein products to meet extremely stringent food-grade and pharmaceutical-grade quality and safety standards. Second, cost issues due to limited carbon source utilization. Traditional Pichia pastoris fermentation is highly dependent on glycerol during the biomass accumulation stage. The supply and price of glycerol are subject to drastic fluctuations in the international bulk oil and bioenergy markets. In contrast, sucrose or molasses (a byproduct of the sugar industry) is a high-quality carbon source that is produced in large quantities, inexpensive, and readily available. However, wild-type Pichia pastoris lacks invertase and cannot directly utilize sucrose, resulting in this highly cost-effective agricultural byproduct being unable to be effectively utilized in yeast fermentation for a long time.

[0003] To address the risk of antibiotic-free gene residue, researchers both domestically and internationally have developed several antibiotic-free screening systems, such as auxotrophic complementation systems based on HIS and URA3, and carbon source utilization markers using GUT1. However, these systems often suffer from drawbacks such as high screening background, low gene knockout or integration efficiency, and susceptibility to interference from complex intracellular carbon metabolism repression effects, making it difficult to maintain long-term genetic stability in large-scale industrial stainless steel fermenters. Therefore, developing a novel, integrated platform that combines excellent antibiotic-free screening capabilities with efficient utilization of inexpensive carbon sources is an urgent need for the green biomanufacturing of eukaryotic recombinant proteins. Summary of the Invention

[0004] Technical issues The technical problem to be solved by this invention is the lack of a recombinant yeast screening system that combines excellent "antibiotic-free screening function" and "efficient utilization of inexpensive carbon source".

[0005] Technical solution This invention provides a sucrose hydrolase based on Aspergillus flavus. SucF A bifunctional selectable marker screening system for Pichia pastoris. This system can not only completely replace antibiotic resistance selectable markers, but also endow Pichia pastoris with the ability to directly and efficiently catalyze the hydrolysis of high-concentration sucrose, significantly reducing the cost of industrial fermentation.

[0006] This invention provides a sucrose hydrolase derived from Aspergillus flavus. SucF The expression box, the expression box is pTEF-IN- SucF The expression box is driven by the heterogeneous constitutive promoter pTEF-IN. SucF The pTEF-IN nucleotide sequence is shown in SEQ ID NO:1. SucF The sequence is shown in SEQ ID NO:2, and its composition includes bases 633-2159 of sequence number XM_041292761.2, with the sequence front combination. Suc2 Signal peptide. In some embodiments of the present invention, one, two, or more codons in SEQ ID NO:2 are mutated, resulting in the encoded signal peptide. SucF Mutations occur at the following sites: T65A, N157I, R322Q, S323G, T65A / R322Q, or T65A / R322Q / S323G.

[0007] This invention provides a product containing the pTEF-IN- SucF The recombinant expression vector for the expression cassette can use pPIC3.5K, pGAPZ, pPIC9K, or pPICZα as the backbone vector for integration of pTEF-IN- into Pichia pastoris GS115, X33, SMD1168, or KM71. SucF Expression box.

[0008] This invention provides the pTEF-IN- SucF Application of expression frames in the construction of recombinant Pichia pastoris, the pTEF-IN- SucF After the expression cassette was transferred into *Pichia pastoris*, the resulting recombinant *Pichia pastoris* was able to utilize 10-40 g / L sucrose as the sole carbon source. When performing gene insertion, knockout, or replacement on the recombinant *Pichia pastoris*, positive clones could be screened on antibiotic-free culture media using sucrose as the sole carbon source. Furthermore, the recombinant *Pichia pastoris* could efficiently produce the target compound using 10-40 g / L (especially 40 g / L) sucrose as the carbon source. The *Pichia pastoris* species include X33, SMD1168, and KM71, as well as the auxotrophic GS115.

[0009] In some embodiments of the present invention, the pTEF-IN- SucFWhen expression frames are used as bifunctional selection markers to screen positive clones that have undergone gene insertion, the main steps include: (1) Using pTEF-IN- SucF Expression cassettes and homologous arm sequences for gene insertion are constructed to obtain pTEF-IN- SucF Gene editing donor vector for expression cassettes; (2) The gene editing donor vector constructed in step (1) is introduced into the host cell, so that the pTEF-IN- SucF The expression cassette undergoes homologous recombination with the host cell genome, achieving targeted insertion of the target gene; (3) The host cells obtained in step (2) are cultured in a selective medium containing sucrose, in which correct gene insertion occurs and functional expression occurs. SucF Positive clones were able to grow using sucrose as a carbon source without insertion or... SucF Cells that do not express the correct expression cannot survive in the selective culture medium; (4) Select single clones that have grown well on sucrose selective medium in step (3) and perform PCR and / or sequencing verification to confirm that the target gene has been correctly inserted into the target position of the genome.

[0010] Preferably, the selective culture medium in step (3) is sucrose selection medium (YPS) with sucrose as the sole carbon source.

[0011] In some embodiments of the present invention, the pTEF-IN- SucF When expression frames are used as bifunctional selection markers to screen for positive clones that have undergone gene knockout, the main steps include: (1) Construct a knockout plasmid for gene knockout, wherein the knockout plasmid contains the pTEF-IN- SucF Expression cassettes, corresponding to the sgRNA and Cas9 expression cassettes of the target knockout gene, and homologous arm sequences upstream and downstream of the target knockout gene locus were prepared as donor fragments. (2) The knockout plasmid and donor fragment constructed in step (1) are introduced into the host cell, and the host cells that have successfully introduced the plasmid are screened by sucrose selective medium, and the CRISPR gene editing system is activated; the donor fragment is integrated into the target knockout site of the genome through homologous recombination to achieve the knockout of the target gene; (3) The host cells obtained in step (2) are cultured in a sucrose-selective medium. Positive clones that have undergone correct homologous recombination and whose target gene has been successfully knocked out can grow normally using sucrose as the sole carbon source, while those without pTEF-IN- SucF Cells in the expression cassette cannot survive in selective culture media; (4) Select single clones that grow well on sucrose selective medium in step (3) and further verify them by PCR amplification or DNA sequencing to confirm that the target gene has been accurately knocked out and there is no unexpected random integration.

[0012] Preferably, the sucrose selective medium in step (3) is a sucrose selection medium (YPS) with sucrose as the sole carbon source.

[0013] In some embodiments of the present invention, the pTEF-IN- SucF When expression frames are used as bifunctional selection markers to screen positive clones that have undergone gene substitution, the main steps include: (1) Constructing a plasmid for gene substitution, the plasmid containing expression Cas9 Protein expression cassettes, sgRNAs targeting the gene knockout loci, and pTEF-IN- for screening. SucF The expression frame; the plasmid further includes a donor fragment, which is composed of an upstream homologous arm of the locus to be knocked out, a foreign target gene, and a downstream homologous arm connected in series; wherein the foreign target gene is located between the upstream and downstream homologous arms and is used to replace the corresponding original target gene in the genome.

[0014] (2) The replacement plasmid and donor fragment constructed in step (1) are introduced into the host cell, and the host cells that have successfully introduced the plasmid are screened by sucrose selective medium to activate the CRISPR gene editing system; the donor fragment is integrated into the target knockout site of the genome through homologous recombination to achieve the precise replacement of the target gene by the target gene; (3) The host cells obtained in step (2) are cultured in a sucrose-selective medium, wherein positive clones that have undergone correct homologous recombination and whose target gene has been successfully replaced can grow normally using sucrose as the sole carbon source, while those without pTEF-IN- SucF Cells in the expression cassette cannot survive in selective culture media; (4) Select single clones that grow well on sucrose selective medium in step (3) and further verify them by PCR amplification, Southern blotting and / or DNA sequencing to confirm that the original target gene has been accurately replaced with the target replacement gene.

[0015] Preferably, the exogenous target gene includes a reporter gene, an resistance marker gene, a mutant gene, or a heterologous gene.

[0016] Preferably, the selective culture medium in step (3) is sucrose selection medium (YPS) with sucrose as the sole carbon source.

[0017] In some embodiments of the present invention, the pTEF-IN-SucF Expression cassettes and histidine auxotrophic markers are combined to construct a dual-marker composite screening system. The construction of high-expression Pichia pastoris hosts typically involves multiple rounds of gene editing, including metabolic pathway optimization and multi-copy integration of exogenous genes. Using traditional resistance markers (such as Zeocin and G418) not only leads to marker conflicts during multiple editing rounds but also poses biosafety risks. Therefore, it is necessary to develop a reusable "antibiotic-free" screening system that does not require the introduction of resistance genes. The dual-marker strategy proposed in this invention does not rely on any antibiotic resistance genes and combines the advantages of "antibiotic-free screening, retrievable markers, and high-precision editing." It is particularly suitable for most Pichia pastoris recombinant expression systems based on commonly used strains such as GS115, KM71, and X-33. It can support the sequential completion of multiple editing steps, such as multi-copy integration and metabolic pathway optimization, ultimately obtaining a safe, efficient, and antibiotic-free production strain.

[0018] In some embodiments of the present invention, the gene insertion is the insertion of the coding gene for human lactoferrin, human osteopontin, or other recombinant milk proteins into the pPIC9k expression plasmid.

[0019] This invention provides a product containing the pTEF-IN- SucF The recombinant Pichia pastoris expressing the expression cassette was used with GS115, X33, SMD1168, and KM71 as hosts, and pPIC3.5K, pGAPZ, pPIC9K, and pPICZα as vectors. The original resistance gene in the vector was replaced with pTEF-IN- SucF Expression box.

[0020] This invention provides recombinant Pichia pastoris for the production of recombinant proteins, using GS115, X33, SMD1168, or KM71 as the host and pPIC3.5K, pGAPZ, pPIC9K, or pPICZα as the vector, replacing the original resistance gene on the vector with pTEF-IN- SucF An expression cassette is constructed, and the encoding genes for human lactoferrin or human osteopontin are linked to the vector. In some embodiments of the present invention, the recombinant Pichia pastoris also expresses chaperone proteins that facilitate protein expression, such as HAC1, PDI, PAB1, SEC12p, SEC13p, and SEC16p.

[0021] This invention provides a method for producing recombinant proteins using the recombinant Pichia pastoris described above. The method employs a two-stage fermentation process: during the cell growth stage, inexpensive sucrose is used as the initial carbon source for biomass accumulation without the addition of any antibiotics; after the sucrose is depleted, the induction stage is initiated, and methanol is added to induce the secretion and expression of exogenous recombinant proteins.

[0022] In some embodiments of the present invention, the main steps include: (1) The recombinant Pichia pastoris was inoculated into YPD liquid medium and cultured overnight (about 16-20 hours) at 28-30℃ and 200-250 rpm with shaking until the cells reached the logarithmic phase; (2) The activated seed liquid was prepared according to the initial OD 600 Transfer approximately 0.5-1.0 μL of the culture medium to BMSY medium and incubate at 28-30°C with shaking at 200-250 rpm until the cell density reaches the OD value. 600 Approximately 10-20; (3) Centrifuge to collect bacterial cells, discard the supernatant, resuspend the bacterial cells in BMMY medium, and induce expression by shaking culture at 28-30℃ and 200-250rpm; (4) During the induction period, methanol shall be added every 24 hours until the final concentration is 0.5%-1.0% (v / v); (5) After 120 hours of induction culture, the fermentation broth was collected, and the cells and supernatant were separated by centrifugation. The target protein was recovered from the supernatant or cells.

[0023] Preferably, the BMSY medium is a sucrose fermentation medium, and the BMMY medium is a methanol-induced medium.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention is the first to construct a system based on Aspergillus flavus. SucF The Pichia pastoris antibiotic-free screening system perfectly combines "screening marker function" with "low-cost carbon source utilization". Under 40g / L sucrose pressure, the gene integration efficiency can reach 100%, and the cost is 200-300 times lower than that of traditional antibiotic-mediated screening such as Zeocin.

[0025] (2) Obtained through protein engineering SucF The triple mutant (T65A / R322Q / S323G) significantly improved the microenvironment of the catalytic pocket, increasing the sucrose hydrolysis rate by 57%, providing a highly efficient catalytic element for the rapid conversion of sucrose in industrial high-density continuous fermentation.

[0026] (3) Using histidine-deficient Pichia pastoris GS115 as the host, a “ SucFA dual-labeled antibiotic-free expression system utilizing sucrose and histidine auxotrophic factors was developed. Under antibiotic-free screening conditions, the secretion yields of human lactoferrin (hLF) and human osteopontin (hOPN) reached 60.3 mg / L and 25.5 mg / L, respectively. The produced high-value human milk proteins (hLF and hOPN) were confirmed by SDS-PAGE and Western Blot analysis to have excellent purity, fully compliant molecular weight, and no residual antibiotic genes, meeting the safety standards for green biomanufacturing and food-grade and pharmaceutical-grade end products, and possessing enormous potential for industrial substitution. Attached Figure Description

[0027] Figure 1 Screening of sucrose hydrolases and their suitable promoters. A: Plate screening of sucrose hydrolases; B: Host growth under different sucrose concentrations; C: Adaptors SucF Promoter selection for expression systems.

[0028] Figure 2 A map of plasmids for gene editing systems.

[0029] Figure 3 Validation of gene integration and knockout efficiency. A: Validation of genome integration efficiency; B: SucF C: Validation of the optimal screening concentration, compared with the knockout efficiency of traditional resistance;

[0030] Figure 4 SucF Screening results for single-point mutations and combined mutations.

[0031] Figure 5 Screening for regulatory factors of antibiotic-free expression of human lactoferrin and osteopontin.

[0032] Figure 6 SDS-PAGE and Western Blot were used to verify the expression of human lactoferrin and osteopontin. A: SDS-PAGE verification of human lactoferrin; B: Western Blot verification of human lactoferrin; C: SDS-PAGE verification of human osteopontin; D: Western Blot verification of human osteopontin. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below through specific embodiments. Unless otherwise specified, the conventional experimental techniques used in the embodiments are routine operations in molecular biology and microbial fermentation engineering.

[0034] 1. The hosts and plasmids used in this invention include: Pichia pastoris strains X33 and GS115, purchased from Invitrogen. The vector pGAPZA is a commercially available Pichia pastoris expression plasmid, purchased from Invitrogen. The vector pPIC9K is a commercially available Pichia pastoris expression plasmid, purchased from Invitrogen.

[0035] 2. Sucrose selection medium (YPS plate): composed of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L agar. It is sterilized by steaming at 118°C for 20 min, and the final concentration of 40 g / L sucrose is filtered through a 0.2 mm filter membrane for sterilization.

[0036] Auxotrophic antibiotic-free selection medium (MD–HIS / sucrose plate): 20 g / L-40 g / L sucrose, 1.34 g / L amino acid-free yeast nitrogen source (YNB), 4 × 10 -5 % (w / v) biotin and 20 g / L agar.

[0037] Sucrose fermentation medium (BMSY): 5g yeast extract and 10g peptone were added to 349 mL distilled water. After steam sterilization at 118℃ for 20 min, 50 mL of 13.4% yeast nitrogen source, 50 mL of 1M pH 6.0 potassium phosphate buffer, and 1 mL of 0.02% biotin were added under aseptic conditions. The sucrose concentration of 40g / L was then filtered through a 0.2 mm filter membrane for sterilization.

[0038] The methanol-induced secretory expression medium (BMMY) for the second stage is formulated as follows: 5 g yeast extract, 10 g peptone, 349 mL distilled water, steam sterilized at 118 °C for 20 min, then 50 mL of 13.4% yeast nitrogen source, 50 mL of 1M pH 6.0 potassium phosphate buffer, 1 mL of 0.02% biotin, and 5 mL of sterile methanol are added. After entering the induction period, pure methanol is added every 24 hours until the final concentration is 15 g / L.

[0039] Method for detecting recombinant human lactoferrin: After centrifugation to remove cell debris and impurities, the supernatant was collected for analysis of recombinant lactoferrin and osteopontin expression. First, SDS-PAGE was performed to preliminarily assess the titer range of recombinant human lactoferrin to guide appropriate sample dilution and ensure the detection falls within the analytical linear range. For quantification, 100 μL of standard and diluted sample were aliquoted and incubated at 37°C for 90 minutes. After discarding the liquid, 100 μL of biotinylated antibody working solution was added, and incubation continued at 37°C for 60 minutes. After washing four times with washing buffer, 100 μL of streptavidin conjugate working solution was added, and incubation was carried out at 37°C for 30 minutes. After washing four more times, 90 μL of substrate reagent and 50 μL of stop solution were added sequentially, and incubation was carried out at 37°C for 15 minutes. The absorbance was measured at 450 nm, and the concentration of recombinant protein in the sample was calculated by interpolation using a standard curve.

[0040] Electroporation and Validation Methods for Pichia pastoris: Using a correctly sequenced plasmid line as a template, the plasmid was digested with restriction endonucleases to form a linearized vector. The linearized fragment was purified using a kit. 80 μL of competent Pichia pastoris cells were mixed thoroughly with 10 μL of linearized recombinant plasmid DNA and transferred to a 0.2 cm electroporation cuvette preheated to -20°C. The cuvette containing the mixture was then incubated on ice for 5 min. The electroporator parameters were adjusted to the Pichia pastoris setting, voltage 1.5 kV, time approximately 5 ms. After electroporation, 1 ml of pre-chilled 1 M sorbitol solution was quickly added to the cuvette, gently mixed, and the mixture was transferred to a centrifuge tube. The mixture was incubated at 30°C for 1–2 h, followed by centrifugation at 3000 rpm for 5 min. Discard 800 μl of supernatant, blow the remaining bacterial cells evenly, spread them on YDS or corresponding antibiotic plates, and incubate at 30°C for 2-4 days until single colonies grow. Verify the correctness by colony PCR.

[0041] Example 1: Screening of sucrose hydrolases and suitable promoters To construct a screening platform that does not rely on conventional antibiotics, the classic Saccharomyces cerevisiae invertase gene was used. Suc2 Using EnzymeMiner (https: / / loschmidt.chemi.muni.cz / enzymeminer / ) as a probe, extensive homology comparisons were performed in the fungal genome to screen four representative candidate invertase genes for heterologous host compatibility testing in Pichia pastoris: genes derived from Saccharomyces cerevisiae. Suc2 (Serial number AB534225.1), source of Aspergillus flavus SucF (The sequence of bases 633-2159 of serial number XM_041292761.2), derived from Aspergillus niger. SucN (Serial number XM_001393172.3) and Bacillus subtilis-derived...SacB (Serial number MH178235.1). All gene fragments were synthesized in their entirety by Suzhou Genewise Biotech Co., Ltd.

[0042] Using the Pichia pastoris constitutive expression vector pGAPZA as the basic backbone, the above four genes were inserted into the target plasmid. Eco RI and Not Between the two restriction enzyme sites, construct the recombinant plasmid pGAPZA- Suc2 (pGAPS2A), pGAPZA- SucF (pGAPSFA), pGAPZA- SucN (pGAPSNA) and pGAPZA- SacB (pGAPSBA), the above plasmid was synthesized by GENEWIZ Biotechnology Co., Ltd. Wild-type Pichia pastoris X33, which cannot naturally utilize sucrose, was used as the initial host. Avr After digestion with enzyme II, the above plasmids were electroporated to obtain recombinant engineered bacteria Su1, Su2, Su3, and Su4, respectively. The recombinant engineered bacteria and the empty vector strain were inoculated onto YPS solid plates with 40 g / L sucrose as the sole carbon source and cultured at 30°C for 48 h. Figure 1 A). The results showed that Su1 (expressing fungal-derived invertase) Suc2 ), Su2 ( SucF ), Su3 ( SucN ) produces plump single colonies. And when transformed into a prokaryotic source... SacB The Su4 strain exhibited extremely sparse and stunted growth, making it unsuitable as a positive marker for antibiotic-free selection. Further testing was conducted on the growth of different recombinant engineered strains at continuous sucrose concentration gradients from 5 g / L to 60 g / L. Figure 1 B). The results showed that Su2 exhibited a higher growth rate at sucrose concentrations above 10 g / L, and still maintained a growth advantage at a sucrose concentration of 40 g / L. Therefore... SucF They were selected as subjects for subsequent experiments.

[0043] Given the conventional constitutive promoter of the pGAPZA plasmid vector GAP It is commonly used to drive the optimization of exogenous protein expression and is not suitable for driving selection markers. Therefore, we intend to screen a set of suitable markers. SucF The promoter for expression was determined. Using pGAPZA as a template, the vector fragment for the promoter selection plasmid was amplified using pGAPZA-ZT-F and pGAPZA-ZT-R primers; using pGAPSFA as a template, pGAP-... SucF -F、pGAP- SucF -R indicates primer amplification to obtain the desired result. GAP promoter SucFExpression cassette. The final plasmid pGAP-SFA was constructed using Gibson assembly. Primers P-ZT-F, P-ZT-R, etc., were selected using the promoters listed in Table 1. SucF The GAP promoter sequence in the expression cassette was replaced with promoters FLD, GCW, DAS, and TEF-IN, ultimately constructing plasmids pFLD-SFA, pGCW-SFA, pDAS-SFA, pTEF-IN-SFA, and the control pGAP-SFA. Natural Pichia pastoris X33 was used as the initial host. Avr Following enzyme digestion (II), the five promoter groups were electroporated to screen plasmids. The electroporated engineered bacteria were then inoculated onto YPS agar plates with 20 g / L sucrose as the sole carbon source and cultured at 30°C for 48 h to obtain recombinant engineered bacteria Su5, Su6, Su7, Su8, and Su9. Further processing was performed using… SucF -Verif-F、 SucF Verif-R amplifies the target gene fragment; colonies that amplify the expected band are considered positive. The positive rate is calculated as (number of colonies positive for the screening signal peptide / number of colonies positive for the GAP signal peptide) × 100%, directly reflecting the efficiency of the transformation experiment. Results showed that the transformation rate of Su8-positive recombinants was as high as 89%. Therefore, pTEF-IN was established as... SucF The promoter of the expression cassette. pTEF-IN-SFA plasmid. SucF The sequence of the expression box is SEQ ID NO:3.

[0044] Table 1: Primers for screening sucrose hydrolase promoters

[0045] Example 2: Application of pTEF-IN- SucF Gene insertion and gene knockout of expression frames (1) Gene insertion This embodiment first confirms the pTEF-IN- SucF Expression frame selection markers can truly replace traditional antibiotic resistance genes for routine genome insertion manipulation because traditional antibiotic plasmids integrate into the AOX1, GAP, and HIS sites. This example verifies pTEF-IN- SucF Whether the expression frame is applicable at different integration sites. Using the pTEF-IN-SFA constructed in Example 1 as a template, pPICZαA- SucF -PD-F、pPICZαA- SucF -PD-R is used as a primer to amplify pTEF-IN- SucF Expression box; using pPICZαA as a template, pPICZαA- SucF -ZT-F,pPICZαA- SucF-ZT-R is the primer amplification vector fragment, pTEF-IN- SucF pTEFSFA- was constructed by fusing expression frames and vector fragments. AOX1 Plasmids. (See Table 2 for details.) HIS - SucF -ZT-F and other primer series will be used to integrate the core classic integration site ( GAP , HIS Insert pTEFSFA- AOX1 The front end of the AOX1 promoter plasmid was used to obtain the knock-in plasmid pTEFSFA- HIS pTEFSFA- GAP ( Figure 2 Use them separately. Sac I, Avr II. Sal The plasmid was linearized by single-enzyme digestion with restriction endonuclease I, electrotransformed into wild-type host bacteria X33, and plated on 40 g / L sucrose solid plates. The genome integration efficiency was verified by the number of positive clones on the plates. SucF -Verif-F、 SucF -Verif-R, as identified by PCR, achieved a genome integration efficiency of 85% at a sucrose concentration of 20 g / L. Figure 3 A). Integration efficiency is defined as the proportion of colonies successfully integrated by PCR to the total number of clones on the plate.

[0046] (2) Gene knockout To verify pTEF-IN- SucF Can expression frame selection markers truly replace traditional antibiotic resistance genes for routine genome knockout manipulation? Using pPpT4 plasmid as a template, pPpT4- SucF -ZT-F,pPpT4- SucF -ZT-R is the primer for amplifying the vector fragment; pTEF-INSFA is used as the template, and primer pPpT4- SucF -PD-F, pPpT4- SucF -PD-R amplification yields pTEF-IN- SucF Expression frame, linking the vector fragment with pTEF-IN- SucF Expression cassette fusion to replace bleomycin resistance in the pPpT4 plasmid with pTEF-IN- SucF The expression frame was used to obtain the knockout plasmid pPpT4F.

[0047] The core gene Ku70, which regulates non-homologous end ligation repair, was knocked out using a CRISPR-Cas9 system mediated by the pPpT4F plasmid. The sgRNA was designed using the ChopChop online tool (https: / / chopchop.cbu.uio.no / ). The fusion fragment used to construct the Ku70 knockout plasmid was obtained by overlap extension PCR using Ku70-AF, Ku70-BF, CR, DR, and ER primers. The fusion fragment contained Ku70 sgRNA, a hammerhead ribozyme, and a hepatitis D virus ribozyme. The vector fragment was obtained by PCR amplification using pPpT4F as a template and pPpT4-ZT-F and pPpT4-ZT-R primers. The fusion fragment and the vector fragment were further ligated by overlap extension PCR to complete the construction of the knockout plasmid pPpT4F-Ku70. Homologous recombination donor fragments were obtained using the X33 genome as a template via primers Ku70-UP-F, Ku70-UP-R, Ku70-DN-F, and Ku70-DN-R, and then fused via overlap extension PCR. After electroporation of X33 competent cells with pPpT4F-Ku70 and the homologous recombination donor fragments, the fragments were plated on 20 g / L sucrose agar plates. Colony PCR using primers Ku70-Verif-F and Ku70-Verif-R confirmed a knockout positivity rate of 10%–20% (knockout positivity rate refers to the proportion of colonies confirmed by PCR to have successfully knocked out the gene out of the total number of clones on the plate). This knockout positivity rate is consistent with the screening efficiency using traditional antibiotics. Figure 3 B). It is evident that, based on Aspergillus flavus... SucF Sucrose-based markers are fully capable of serving as a highly efficient and versatile non-antibiotic gene engineering manipulator element, applicable to gene editing in Pichia pastoris. Screening experiments with different sucrose concentrations revealed that gene integration rate reached 100% under a sucrose concentration of 40 g / L. Figure 3 C).

[0048] Table 2: Primers required for constructing gene editing systems

[0049] Example 3: Semi-rational computer-aided design of highly catalytically active catalysts SucF mutant The foregoing examples verified the wild-type Aspergillus flavus. SucF While it can be considered as a screening marker, its natural catalytic rate remains insufficient under the pressure of high-sugar substrates in modern industrial fermentation. This example targets the wild type. SucF Implement data-driven, computer-based semi-rational engineering reforms. First, […]. SucFThe full-length amino acid sequences were submitted to the EV Coupling online server. Multiple sequence alignment of fungal homologous invertase sequences was used to calculate co-evolutionary coupling scores between residue sites, and positive effect variant sites with evolutionary scores >2.0 were screened. Next, the DeepDDG neural network server was used to predict the single-point mutation folding free energy change (ΔΔG) of candidate sites, retaining only amino acid substitutions with ΔΔG > 0. Dynamic spatial backbone scanning was performed using HotSpotWizard 3.0 to identify highly flexible and non-absolutely conserved catalytic periphery residues located near substrate-binding pockets and hydrolysis product efflux channels. Based on the above analyses, a total of 19 candidate residues were screened.

[0050] pGAPZA- SucF Using the template, corresponding single-point mutant plasmids were constructed through site-directed mutagenesis. Primers are shown in Table 3. Successfully sequenced mutants were then... AvrII After being digested into linear fragments, the fragments were electroporated into X33 and inoculated into YPS medium containing 40 g / L sucrose for fermentation for 48 h. The residual sucrose content in the fermentation broth was measured as a screening indicator. The results showed that the single-point mutant plasmid pGAPZA- SucF- T65A, pGAPZA- SucF- N157I, pGAPZA- SucF- R322Q and pGAPZA- SucF- S323G was consumed by 40 g / L sucrose to only 7.2, 8.5, 7.9, and 8.0 g / L respectively within 48 hours. Figure 4 A).

[0051] To explore the spatial synergistic and complementary effects among mutation sites, iterative multi-site combinations were constructed from the four high-value sites mentioned above, resulting in a series of multi-site composite mutants. The results showed that the pGAPZA- mutant exhibited the highest transformation activity among all the combined mutants. SucF- The T65A / R322Q / S323G mutant, with a residual sucrose concentration of 5.5 g / L, exhibited a 57% higher sucrose hydrolysis conversion rate compared to the wild-type (12.9 g / L) and a 13% higher rate than the best-performing single-site mutant. The double mutant T65A / R322Q also showed a 51% improvement over the wild-type. The four-site mutant (pGAPZA-)... SucF- The catalytic activity of T65A / N157I / R322Q / S323G actually decreased, indicating that excessive mutations disrupted the precise grid of the enzyme's active center. Figure 4 B). It can be seen that the improved pGAPZA- SucF- The T65A / R322Q / S323G expression cassette encodes highly active... SucF Mutants can be used as selection markers and can also enable engineered bacteria to utilize sucrose as a carbon source.

[0052] Table 3: Primers required for mutant construction

[0053] Example 4: pTEF-IN- SucF Application of mutant expression cassettes in constructing antibiotic-free engineered bacteria that produce high-value-added milk proteins This embodiment aims to apply the constructed high-performance antibiotic-free screening marker element to the industrial-scale green manufacturing of high-value human lactoferrin and human osteopontin. First, fully codon-optimized human lactoferrin is processed using pPIC9k series plasmids.<SEQ ID NO:4> or human osteopontin<SEQ ID NO:5> The expression cassette was introduced into Pichia pastoris GS115, and recombinant expression lines hLFC and hOPNC were obtained by screening on histidine-free MD-HIS solid plates.

[0054] To further optimize the expression level of the recombinant protein, a vector fragment was amplified using pGAPZA as a template and pGAP-SF-Zeo-ZT-F and pGAP-SF-Zeo-ZT-R as primers. The vector fragment was an expression framework with bleomycin resistance removed. The three-point mutant plasmid pGAPZA- SucF Using T65A / R322Q / S323G as a template and pTEF-SF-ZEO-PD-F and pTEF-SF-ZEO-PD-R as primers, pTEF-IN- is amplified. SucF The mutant expression cassette was assembled using Gibson to obtain the plasmid pGXSFA, which replaced the bleomycin resistance of pGAPZA with the SucF ​​mutant expression cassette. Then, the recombinant expression lines hLFC and hOPNC were modified to enhance transcription and promote protein processing and transport, respectively. HAC1 , PDI , PAB1 , SEC12P , SEC13P , SEC16P After inserting the GAP promoter into plasmid pGXSFA, pGXSFA- was constructed. HAC1 pGXSFA- PAB1 pGXSFA- PDI pGXSFA- SEC12p pGXSFA- SEC13p and pGXSFA- SEC16p The required primers are listed in Table 4. These chaperone plasmids... Avr After digestion with enzyme II, the enzymes were electroporated into hLFC and hOPNC, respectively, and plated on YPS solid plates with 40 g / L sucrose as the sole carbon source to obtain the corresponding expression strains. Finally, using " SucFThe dual dominant complementary screening constructed by "sucrose utilization marker" and "histidine defective marker" can obtain highly stable recombinant antibiotic-free strains with multi-gene co-integration.

[0055] The recombinant protein expression level of the antibiotic-free strain was further verified by shake-flask assay. The recombinant antibiotic-free strain was inoculated into 50 mL of BMSY medium and cultured at 30°C and 220 rpm. The first stage (0-24 h) was the cell growth stage, and the second stage (24-120 h) was the induction stage. During the induction stage, after centrifugation and washing, the cells were replaced with BMMY medium, and 1.5% (v / v) pure methanol was added every 24 h. After 120 h of fermentation, the clear supernatant was collected and analyzed by high-sensitivity ELISA and quantitative analysis. Figure 5 It was confirmed that after overexpression of HAC1, the expression level of human lactoferrin increased from 41.1 mg / L to 60.3 mg / L, and the expression level of human osteopontin increased from 15.1 mg / L to 25.5 mg / L. The harvested crude fermentation broth was purified by two-step chromatographic processes and then subjected to SDS-PAGE and specific Western blotting for detection. Figure 6 The electrophoretic pattern showed clear bands at approximately 78 kDa and 36 kDa. The entire process was conducted without the addition of any antibiotics, demonstrating the promising future of green industrial biomanufacturing.

[0056] Table 4: Primers required for constructing pGXSFA series helper gene expression plasmids

[0057] sequence list <SEQ ID NO:1> pTEF-IN <SEQ ID NO:2> SucF <SEQ ID NO:3> pTEF-IN- SucF <SEQ ID NO:4> Human lactoferrin gene <SEQ ID NO:5> Human osteopontin gene.

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Sucrose hydrolase SucF The expression box is characterized by, The expression box is driven by the heterogeneous constitutive promoter pTEF-IN. SucF or SucF mutant, the SucF A mutant is a gene shown in SEQ ID NO:2 whose one, two, or more codons are mutated, resulting in a mutation that encodes a different gene. SucF The mutation occurs, namely: T65A, N157I, R322Q, S323G, T65A / R322Q or T65A / R322Q / S323G.

2. Containing the pTEF-IN- of claim 1 SucF The recombinant expression carrier of the expression frame is characterized by, pPIC3.5K, pGAPZA, pPIC9K, or pPICZαA were used as the backbone vectors.

3. The pTEF-IN- as described in claim 1 SucF The application of expression frames in the construction of recombinant Pichia pastoris is characterized by, The pTEF-IN- SucF After the expression cassette is transferred into Pichia pastoris, the resulting recombinant Pichia pastoris can utilize 10-40 g / L sucrose as the sole carbon source. When gene insertion, gene knockout, or gene replacement is performed on the recombinant Pichia pastoris, positive clones can be screened on antibiotic-free culture medium using sucrose as the sole carbon source as a selection marker. Furthermore, the recombinant Pichia pastoris can generate the target substance using 10-40 g / L sucrose as the carbon source.

4. The pTEF-IN- according to claim 3 SucF The application of expression frames in the construction of recombinant Pichia pastoris is characterized by, The Pichia pastoris includes: X33, SMD1168, KM71 or auxotrophic GS115.

5. The pTEF-IN- according to claim 3 SucF The application of expression frames in the construction of recombinant Pichia pastoris is characterized by, The auxotrophic GS115 is a histidine auxotroph.

6. The pTEF-IN- according to claim 3 SucF The application of expression frames in the construction of recombinant Pichia pastoris is characterized by, The gene insertion refers to the insertion of the encoding gene for human lactoferrin, human osteopontin, or other recombinant milk proteins.

7. Containing the pTEF-IN- as described in claim 1 SucF Recombinant Pichia pastoris with expression frames, characterized in that, The host is GS115, X33, SMD1168, or KM71, and the vector is pPIC3.5K, pGAPZA, pPIC9K, or pPICZαA. The original resistance gene in the vector is replaced with pTEF-IN- SucF Expression box.

8. The recombinant Pichia pastoris according to claim 7, characterized in that, The coding genes for human lactoferrin or human osteopontin are linked to the vector.

9. The use of the recombinant Pichia pastoris according to claim 7 or 8 in the production of recombinant proteins.

10. Sucrose hydrolase SucF Mutant, characterized by, Mutating one, two, or more codons of the gene shown in SEQ ID NO:2, resulting in the encoded... SucF The mutation occurs, namely: T65A, N157I, R322Q, S323G, T65A / R322Q or T65A / R322Q / S323G.

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