Pichia pastoris engineering bacteria with high expression of grass carp-derived antibacterial protein lect2-b and fermentation process thereof

Abstract

This invention discloses an engineered Pichia pastoris strain that highly expresses the grass carp-derived antimicrobial protein LECT2-b and its fermentation process. The strain was constructed by integrating an optimized gene into the Pichia pastoris KM71 genome. The fermentation process was systematically optimized, and after 72 h of induction at a cell density of 400 g / L, the LECT2-b content in the fermentation supernatant reached 2.2 g / L. This method, through secretory expression, allows direct detection of the target protein in the fermentation supernatant, effectively reducing the content of impurity proteins and simplifying the production process. Compared to E. coli expression vectors, it avoids the cumbersome steps of inclusion body fragmentation and renaturation, simplifying the preparation process. The culture medium used is simple to prepare and inexpensive, and the product retains natural broad-spectrum antimicrobial activity. Multiple repeated verifications have been completed in a 2000L fermenter, demonstrating process stability and feasibility for industrial scale-up. It can be used as a feed additive for immune enhancement and disease prevention in aquaculture animals, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to a Pichia pastoris engineered strain that highly expresses the grass carp-derived antimicrobial protein LECT2-b and its fermentation process. Background Technology

[0002] Antimicrobial peptides and small-molecule antimicrobial proteins are a class of small molecules widely found in organisms in nature. They possess positive charges and amphiphilic structures and are important components of the innate immune system. Antimicrobial peptides / proteins exert broad-spectrum antimicrobial activity by disrupting the cell membranes of pathogenic microorganisms, inhibiting both Gram-positive and Gram-negative bacteria, and are less likely to induce drug resistance. They have broad application prospects in medicine, animal husbandry, and aquaculture.

[0003] Leukocyte-derived chemotaxin 2 (LECT2) is a multifunctional cytokine secreted by the liver, involved in biological processes such as regulating liver regeneration, maintaining natural killer T cell homeostasis, and cell carcinogenesis. The novel antimicrobial protein LECT2-b, discovered in the intestines of grass carp, is highly expressed in mucosal immune organs such as the intestines. In addition to possessing the conserved immunomodulatory activities of the LECT2 family, it can also participate in the body's antimicrobial immune response through direct bactericidal activity. LECT2-b can selectively target and kill intestinal pathogens in grass carp, participating in the regulation of intestinal flora homeostasis, and has broad application prospects in aquaculture disease control and other fields.

[0004] Currently, the main technologies for preparing antimicrobial proteins include natural extraction, chemical synthesis, and genetic engineering. Natural extraction is limited by raw material sources, resulting in low yields and complex purification processes, making it unsuitable for large-scale production. Chemical synthesis is costly and primarily used in research, making industrial production difficult. In contrast, using genetic engineering to modify microorganisms to synthesize target antimicrobial proteins not only effectively preserves the protein's bioactivity but also has lower production costs, making it suitable for large-scale industrial production. Currently, several active proteins have achieved large-scale production using this technology.

[0005] Commonly used protein expression systems in genetic engineering include the *E. coli* expression system, the *Bacillus subtilis* expression system, and the *Pichia pastoris* expression system. The *E. coli* system has a clear genetic background, is easy to operate, and produces high expression levels, but it lacks eukaryotic post-translational modification capabilities, and exogenous proteins easily form inactive inclusion bodies, posing a risk of endotoxin contamination. For example, existing technology (CN113061176A) has attempted to express grass carp LECT2 protein in *E. coli*, but the product mostly exists in inclusion body form, requiring cumbersome steps such as denaturation and renaturation to obtain a small amount of soluble active protein, resulting in low purification efficiency and high levels of impurities, which is unfavorable for large-scale production and application. The *Bacillus subtilis* system has good secretory expression capabilities, but its plasmid stability is poor, and the expression product is easily degraded by extracellular proteases. In contrast, the Pichia pastoris expression system combines the rapid growth and ease of high-density fermentation characteristic of prokaryotes with the post-translational modifications of eukaryotes, such as correct protein folding, disulfide bond formation, and glycosylation, which is beneficial for expressing proteins with natural biological activity. Furthermore, this system can achieve secretory expression of exogenous proteins, significantly simplifying subsequent purification processes, and it produces less secreted protein, resulting in high host safety. However, there are currently no reports on the secretory expression of the grass carp-derived antimicrobial protein LECT2-b using Pichia pastoris. Therefore, developing an engineered Pichia pastoris strain capable of efficiently secreting and expressing LECT2-b, along with a stable industrial fermentation process, is of great significance for promoting the practical application of this antimicrobial protein in aquaculture. Summary of the Invention

[0006] This invention addresses the problems of inactive inclusion body formation, cumbersome denaturation and renaturation steps, low purification efficiency, and high impurity protein content when expressing grass carp LECT2-b in *E. coli*. It provides a *Pichia pastoris* engineered strain for high expression of the grass carp-derived antimicrobial protein LECT2-b and its fermentation process. By utilizing a *Pichia pastoris* secretory expression system, the codon-optimized LECT2-b gene is integrated into the *Pichia pastoris* genome, achieving efficient secretory expression of LECT2-b. This invention further optimizes different *Pichia pastoris* strains and key fermentation parameters, establishing optimal expression conditions in shake flasks and fermenters. The *Pichia pastoris* engineered strain constructed in this invention successfully achieved soluble and efficient secretory expression of LECT2-b in both 30L and 2000L fermenters. The product can be directly recovered from the fermentation supernatant without the need for inclusion body renaturation, simplifying the purification process and reducing impurity protein content. The obtained LECT2-b protein retains its natural antimicrobial activity, exhibiting significant inhibitory effects against common intestinal pathogens in grass carp. Furthermore, the process is stable, reproducible, and feasible for industrial scale-up. This invention provides a simple, low-cost fermentation preparation route for grass carp LECT2-b protein, which produces a product with excellent activity. It is of great value for its application in the prevention and control of diseases in aquaculture.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a recombinant Pichia pastoris engineered strain that secretes and expresses the grass carp-derived antimicrobial protein LECT2-b. The engineered strain is obtained by integrating an optimized gene encoding the grass carp-derived antimicrobial protein LECT2-b into the genome of the Pichia pastoris KM71 strain; wherein the nucleotide sequence of the optimized gene is shown in SEQ ID NO.3.

[0008] Furthermore, the optimized gene was integrated into the genome of Pichia pastoris strain KM71 using the pPIC9K vector as a backbone vector.

[0009] Furthermore, the method for constructing the recombinant Pichia pastoris engineered strain includes: (1) The LECT2-b coding sequence shown in SEQ ID NO.1 was optimized according to the codon preference of Pichia pastoris to obtain the optimized gene shown in SEQ ID NO.3; (2) EcoRI and NotI restriction sites were introduced at both ends of the optimized gene, while avoiding the SalI restriction site, and the gene was synthesized into the pPIC9K vector backbone to obtain the recombinant expression plasmid pPIC9K-LECT2-b. (3) Linearize the recombinant expression plasmid using SalI fast digestion enzyme; (4) Preparation of Pichia pastoris KM71 competent cells; (5) The linearized recombinant expression plasmid was transformed into Pichia pastoris KM71 competent cells by electroporation; (6) Positive transformants were obtained through G418 resistance screening; (7) Positive transformants were identified by PCR and sequenced to screen for engineered strains that highly express LECT2-b protein.

[0010] This invention also provides the application of the above-mentioned recombinant Pichia pastoris engineered strain in the high-yield grass carp-derived antimicrobial protein LECT2-b.

[0011] This invention also provides a method for producing grass carp-derived antimicrobial protein LECT2-b by fermentation using the above-mentioned recombinant Pichia pastoris engineered strain, comprising the following steps: (1) Seed culture: The recombinant Pichia pastoris engineered strain was inoculated into seed culture medium and cultured at 25~35℃ and 200~250 rpm until the logarithmic growth phase to obtain fermentation seed liquid; (2) Fermentation tank culture: The fermentation seed liquid was inoculated into the basic culture medium at a volume ratio of 5% to 15%, and glycerol was cultured in batches at 25 to 35°C, pH 5.0 to 6.0, and dissolved oxygen ≥ 30%. (3) Glycerol replenishment: After the glycerol in the basal medium is depleted, glycerol is added at a constant rate of 10~20 g / L / h. (4) Methanol induction: Stop feeding after the wet weight of the cells reaches 200~500 g / L, adjust the temperature to 20~30℃ and the pH to 5.5~6.5, and add methanol at a flow rate of 2~5 ml / h / L for induction. The induction time is more than 72 hours. (5) Collect and purify the supernatant: After induction, centrifuge to collect the fermentation supernatant, and then purify it to obtain grass carp LECT2-b protein.

[0012] Furthermore, the basal culture medium mentioned in step (2) is D'Anjou medium or BSM medium.

[0013] Furthermore, in step (3), the flow rate of the glycerol feed is 15 g / L / h.

[0014] Furthermore, 50% (w / v) glycerin was used for feeding.

[0015] Furthermore, in step (4), feeding is stopped after the wet weight of the bacteria reaches 400 g / L, and methanol induction begins.

[0016] Furthermore, in step (4), the methanol-induced temperature is 28°C, the pH is 6.0, and the methanol flow rate is 3.7 ml / h / L.

[0017] Further, step (5) includes: centrifuging and microfiltration of the fermentation supernatant, collecting the filtrate; separating and purifying the filtrate by passing it through a cation exchange chromatography column; then collecting the eluent, removing salt ions and small molecule impurities by dialysis, and obtaining purified LECT2-b protein.

[0018] Furthermore, the binding buffer used for separating and purifying the filtrate by passing it through a cation exchange chromatography column includes: 3.8 mM Na2HPO4, 16.2 mM NaH2PO4, pH 7.4; the elution buffer includes: 3.8 mM Na2HPO4, 16.2 mM NaH2PO4, 1 M NaCl, pH 7.4.

[0019] The present invention also provides the application of the above-mentioned recombinant Pichia pastoris engineered strain or the above-mentioned method in the preparation of antibacterial feed additives or antibacterial drugs containing grass carp-derived LECT2-b protein.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses the Pichia pastoris secretory expression system to express grass carp antimicrobial protein LECT2-b, which avoids the inclusion body formation problem common in traditional Escherichia coli expression systems. It eliminates the need for cumbersome denaturation and renaturation steps, significantly simplifies the protein purification process, effectively reduces the content of impurities, and improves the purity of the target protein.

[0021] (2) This invention achieves efficient secretory expression of LECT2-b in Pichia pastoris through codon optimization, host strain selection and fermentation conditions. The expression yield is high. After induction at a cell density of 400 g / L for 72 h, the LECT2-b content in the fermentation supernatant reaches 2.2 g / L. Moreover, the obtained LECT2-b protein maintains natural antibacterial biological activity and has a significant inhibitory effect on common pathogens in the intestine of grass carp.

[0022] (3) After the fermentation process established by this invention was successfully verified in a 30L fermenter, it was further verified by three repeated scale-up tests in a 2000L fermenter. The process was stable and reproducible, proving that the method has good feasibility for industrial scale-up and is suitable for large-scale production application.

[0023] (4) The fermentation preparation route provided by the present invention is simple, convenient to operate, and has low production cost. The fermentation product can be directly recovered from the supernatant without complicated downstream processing procedures, which helps to reduce the industrialization threshold.

[0024] (5) This invention fills the technical gap in the secretion and expression of grass carp-derived antimicrobial protein LECT2-b by Pichia pastoris, providing reliable technical support for the promotion and application of this antimicrobial protein in the prevention and control of aquatic diseases, and has high industrial application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a comparison diagram of the LECT2-b codon before and after optimization in Embodiment 1 of the present invention.

[0027] Figure 2 The images show the growth results of Pichia pastoris transformants on YPD plates with a G418 concentration of 2 mg / ml in Example 1 of this invention. The left image shows strain KM71-LECT2-b, and the right image shows strain GS115-LECT2-b.

[0028] Figure 3The results of optimizing the induction conditions in shake flasks in Example 2 of this invention are shown in Figures A and B, which are SDS-PAGE electrophoresis images of the fermentation supernatant and line graphs of LECT2-b concentration in the fermentation supernatant at different induction times, respectively, showing the expression of LECT2-b at six induction time points (0h, 24h, 48h, 72h, 96h, and 120h) and the dynamic change of LECT2-b concentration with induction time. Figures C and D are SDS-PAGE electrophoresis images of the fermentation supernatant and LECT2-b concentration in the fermentation supernatant under different methanol concentrations, respectively. Line plots of LECT2-b concentration were used to compare the expression differences of LECT2-b at four methanol concentrations of 0.5%, 1%, 1.5%, and 2%, as well as the trend of LECT2-b protein concentration with methanol concentration. Figures E and F show SDS-PAGE electrophoresis images of fermentation supernatant and line plots of LECT2-b concentration in fermentation supernatant under different pH conditions, respectively, comparing the expression differences of LECT2-b and the change of LECT2-b protein concentration with pH under four culture conditions of pH 5.5, 6.0, 6.5, and 7.0.

[0029] Figure 4 This is a comparison of two glycerol feeding methods during high-density fermentation in Example 2 of the present invention. Figure A shows the dissolved oxygen (DO) trend under constant-rate feeding. Figure B shows the dissolved oxygen (DO) trend under constant-oxygen feeding. Figure C shows the difference in cell wet weight changes under the two modes.

[0030] Figure 5 This is an example of the effect of initial cell wet weight on protein expression in Example 2 of the present invention. Figure A shows the dynamic changes of cell wet weight under two conditions, and Figure B shows the changes of total protein content in fermentation supernatant at different time points under two conditions.

[0031] Figure 6 This shows the expression of LECT2-b in three different basal culture media (BSM, D'Anjou, FM21) in Example 2 of the present invention.

[0032] Figure 7 This is a graph showing the comparison of expression levels between strains KM71-LECT2-b and GS115-LECT2-b in Example 2 of the present invention.

[0033] Figure 8 Figure 2 shows the identification results of LECT2-b in the fermentation supernatant after 72 hours of induced expression in a fermenter in Example 2 of this invention. Figure A shows the SDS-PAGE verification results of the fermentation supernatant, Figure B shows the SDS-PAGE verification results of the purified LECT2-b, and Figure C shows the results of Western blot detection combined with ImageJ software analysis of the gray values ​​of the protein bands.

[0034] Figure 9 The results of the pilot-scale fermentation of strain KM71-LECT2-b in a 2000L fermenter in Example 2 of this invention are shown.

[0035] Figure 10 The LECT2-b purified in 30L and 2000L fermenters in Example 3 of this invention exhibits antibacterial activity against Aeromonas hydrophila Ah72. Detailed Implementation

[0036] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available. Molecular biology experimental methods in the following embodiments that do not specify specific conditions are all performed under conventional conditions, referring to *Molecular Cloning: A Laboratory Manual*.

[0037] Example 1: Construction of an engineered Pichia pastoris strain expressing the grass carp-derived antimicrobial protein LECT2-b 1. Linearized Pichia pastoris expression plasmid pPIC9K-LECT2-b The original nucleotide sequence of the grass carp-derived antimicrobial protein LECT2-b is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2. To improve its expression efficiency in Pichia pastoris, the LECT2-b coding sequence was codon-optimized according to the codon preference of Pichia pastoris. The optimized nucleotide sequence is shown in SEQ ID NO.3. A comparison of the sequences before and after codon optimization is shown below. Figure 1 As shown.

[0038] EcoRI and NotI restriction endonuclease sites were introduced at both ends of the codon-optimized LECT2-b coding sequence (SEQ ID NO.3), while SalI restriction endonuclease sites were avoided during sequence design to ensure the specificity of subsequent linearization digestion. The designed DNA sequence was synthesized into the pPIC9K vector backbone by Beijing Qingke Biotechnology Co., Ltd., to obtain the Pichia pastoris expression plasmid, named pPIC9K-LECT2-b.

[0039] The pPIC9K-LECT2 plasmid was linearized by digestion with SalI rapid digester to enable rapid integration into the Pichia pastoris genomic DNA via homologous recombination after transformation. The digestion reaction mixture consisted of 20 μg pPIC9K-LECT2-b plasmid, 8 μl SalI rapid digester, 50 μl Quick Cut Buffer, and sterile water to a final volume of 500 μl. The reaction was incubated at 37°C for 1 hour. After the reaction, the linearized plasmid was recovered from the reaction mixture using an agarose gel extraction kit and stored at -20°C for subsequent transformation experiments.

[0040] 2. Preparation of Pichia pastoris KM71 and GS115 competent cells Pichia pastoris strains KM71 and GS115 (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) frozen at -80℃ were thawed on ice, streaked onto YPD solid medium, and incubated at 30℃ for 24 h to obtain single colonies. Single colonies were picked and transferred to 100 ml of YPD liquid medium, activated at 30℃ and 220 rpm for 14 h. The bacterial suspension was then processed according to the following steps to prepare competent cells: a. Centrifuge 20 ml of bacterial suspension at 4℃ and 3000 rpm for 5 min, discarding the supernatant; b. Resuspend the bacterial cells in 30 ml of pre-chilled sterile water, centrifuge, and discard the supernatant; c. Resuspend the bacterial cells in 20 ml of pre-chilled sterile water, centrifuge, and discard the supernatant; d. Resuspend the bacterial cells in 10 ml of pre-chilled sterile 1M sorbitol solution, centrifuge, and discard the supernatant; e. Resuspend the bacterial cells in 1 ml of pre-chilled sterile 1M sorbitol solution, centrifuge, and discard the supernatant; f. Resuspend the bacterial cells in 100 μl of pre-cooled sterile 1M sorbitol solution, and transfer 80 μl to a sterile centrifuge tube to obtain the transformed competent cells. Place the tube on ice for later use.

[0041] 3. Electroconversion Add 20 μl of the linearized plasmid to 80 μl of KM71 and GS115 competent cells respectively, mix well, and then transfer to a 1 mm electroporation cuvette. Incubate on ice for 5 min, followed by electroporation under the following conditions: voltage 2000 V, pulse duration 5 ms, 1 electroporation. Immediately after electroporation, add 600 μl of 1 M sorbitol solution, mix gently, and incubate at 30 °C and 220 rpm for 1 h to recover. After recovery, spread 100 μl of the bacterial culture onto an MD plate and incubate upside down at 30 °C for 2-3 days until single colonies appear.

[0042] 4. Screening of positive clones Pick a single colony from an MD plate and transfer it to a YPD plate with a G418 concentration of 0.5 mg / ml. Incubate at 30°C upside down until a white, round single colony grows. Then transfer it to a YPD plate with a G418 concentration of 2 mg / ml and incubate at 30°C upside down. Figure 2The copy number of LECT2-b in transformants is positively correlated with the concentration of G418. High-copy colonies can be screened by increasing the concentration of G418 in the culture medium.

[0043] Different single colonies were selected and cultured in YPD liquid medium for 12 h. Genomic DNA was then extracted using a yeast genomic DNA extraction kit, and PCR amplification and identification were performed using the genomic DNA as a template. The PCR reaction system consisted of: 20 μl Taq enzyme, 2 μl AOX1-F, 2 μl AOX1-R, 2 μl yeast genomic DNA, and sterile water to a final volume of 40 μl. The PCR reaction conditions were: 94 ℃ pre-denaturation for 5 min; 32 cycles of 95 ℃ for 30 s, 58 ℃ for 30 s, and 72 ℃ for 30 s; 72 ℃ for 7 min; and maintenance at 4 ℃.

[0044] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis, and the integration was determined based on the band size. Bands that matched the theoretical size were cut and recovered, and sent to the company for sequencing identification to confirm the correctness of the LECT2-b gene sequence.

[0045] Example 2: Induction of expression and optimization of fermentation process 1. Shake-flask induced expression method The positive recombinant Pichia pastoris strains (KM71-LECT2-b, GS115-LECT2-b) confirmed by PCR identification and sequencing were used for shake-flask induction of expression according to the following steps: a. Select positive clones and inoculate them into 5 ml of YPD liquid medium containing 100 μg / mL G418 at an inoculation rate of 1% (v / v). Incubate at 28 ℃ and 220 rpm in the dark for 24 h with shaking. b. Take 500 μl of primary culture and inoculate it into a 250 ml Erlenmeyer flask containing 25 mL of BMGY culture medium. Incubate at 28°C and 220 rpm for approximately 16 h, until the bacterial culture reaches OD500. 600 Reaching 3-4; c. Transfer all bacterial culture to 50 ml sterile centrifuge tubes, centrifuge at 4000 r for 5 min at 4°C, collect the bacterial cells and discard the supernatant, then dilute the bacterial cells to OD500 with 100 ml BMMY medium. 600 = 1, then transfer all liquid to a 1L sterile conical flask, seal with 3 layers of sterile gauze, and incubate at 28℃ and 230rpm in a constant temperature shaker. d. Add anhydrous methanol to the bacterial culture at a final concentration of 1% (v / v) every 24 h to induce expression for 96 h; e. Stop the culture, centrifuge at 3000 rpm for 10 min, collect the supernatant for subsequent detection.

[0046] Shake-flask induction conditions optimization method: Single-factor optimization was performed sequentially according to the following three parameters: induction expression time (24 h, 48 h, 72 h, 96 h, 120 h), methanol induction concentration (0.5%, 1.0%, 1.5%, 2.0%, v / v), and induction medium pH (5.5, 6.0, 6.5, and 7.0). The optimization of each parameter was based on the optimal conditions obtained in the previous step, and the culture process was consistent with the preliminary expression process described above. The yield of LECT2-b in shake flasks was determined under the optimal expression conditions.

[0047] Optimization results are as follows Figure 3 As shown, in the induction time optimization experiment, samples were taken at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h for SDS-PAGE analysis and concentration determination. The results showed that the LECT2-b concentration continuously increased from 0 h, reaching a peak at 72 h, and then slightly decreased with increasing time; therefore, the optimal induction time was determined to be 72 h. In the methanol concentration optimization experiment, the expression differences at four methanol concentrations (0.5%, 1.0%, 1.5%, and 2.0%) were compared. Quantitative analysis showed that the LECT2-b concentration reached its peak at 1.0% methanol concentration; concentrations decreased at concentrations below or above 1.0%; therefore, the optimal methanol concentration was determined to be 1.0% (v / v). In the pH optimization experiment, the expression differences under four culture conditions (pH 5.5, 6.0, 6.5, and 7.0) were compared. The results showed that the LECT2-b concentration was highest at pH 6.0; pH values ​​that were too low or too high led to a decrease in concentration; therefore, the optimal induction pH was determined to be 6.0. In summary, the optimal conditions for shake-flask induction of expression are: induction time of 72 h, methanol concentration of 1.0% (v / v), and culture medium pH of 6.0. These conditions will serve as the basis for subsequent fermenter scale-up validation.

[0048] 2. Expression validation and optimization in a 30L fermenter (1) Validation steps for a 30L fermenter a. Inoculate the bacterial culture into two 500ml bottles of YPD medium and activate it at 30℃ and 220rpm for 24 hours to obtain the fermentation seed liquid.

[0049] b. Fermentation preparation: Install dissolved oxygen electrodes in the fermenter and install and calibrate the pH electrode; prepare 10L of BSM basal medium (containing 400g glycerol as a carbon source), fully dissolve it, pour it into the sterilized fermenter, sterilize at 121℃ for 21 minutes, then cool to 30℃. Stir at 200rpm for 6 minutes. 3After 1 hour of aeration, calibrate the dissolved oxygen electrode to ensure dissolved oxygen is 100%. Configure the feeding system, including: 500 ml of 50% (v / v) defoamer, 2 L of 50% (w / v) glycerol (containing 24 ml of PTM1), 500 ml of ammonia, and 2 L of methanol (containing 24 ml of PTM1). After sterilization and cooling, connect the system to the fermenter and adjust the pH to 5.0 automatically.

[0050] c. Inoculate the seed culture (10% of the volume of the basal culture medium) and 45 ml of PTM1 using the flame inoculation method to start fermentation. During fermentation, adjust the speed of the agitator and the aeration rate to maintain dissolved oxygen above 30%.

[0051] d. About 18 hours after fermentation begins, dissolved oxygen rises to 80%, indicating that the glycerol in the basal medium is depleted. Then, supplement with 50% glycerol to increase biomass. The feeding rate should be adjusted to maintain dissolved oxygen at 20%-40%.

[0052] e. About 48 hours after the start of fermentation, feeding was stopped, the wet weight was measured to be 280 g / L, feeding was stopped, the temperature was adjusted to 28℃, the pH was automatically controlled to 6.0, and after starvation for 1 hour, methanol induction was carried out, with a methanol flow rate of 3.7 ml / h / L.

[0053] f. After induction for 3 consecutive days, samples were aseptically taken every 24 hours, the supernatant was collected by centrifugation, and SDS-PAGE gel electrophoresis was performed. After electrophoresis, Coomassie Brilliant Blue was used for staining for 15 minutes, and the protein composition was observed after destaining with boiling water until the background color was clear.

[0054] (2) Optimization methods and results of fermentation conditions Based on the above verification steps, we further optimized three key parameters: glycerol feeding method, wet weight of inducing starting cells, and type of basal culture medium.

[0055] a. Comparison of glycerol fed-batch methods: BSM inorganic salt medium was used as the basic fermentation medium, with the addition of PTM1 microsalt solution. The pH was adjusted to 5.0 with 28% (w / v) ammonia. During fermentation, the DO value was controlled above 20% by increasing the aeration rate and adjusting the fermentation speed. The maximum aeration rate was 6 m³ / s. 3The maximum rotation speed was 800 r / min. Fermentation was carried out in BSM medium containing 4% glycerol. After approximately 20 hours, the dissolved oxygen (DO) level rose sharply, indicating glycerol depletion. At this point, a 50% (w / v) glycerol feed (containing 12 ml of PTM1 microsalt solution per liter) was added. Two feeding methods were compared: constant dissolved oxygen feeding (DO-stat feeding) and constant flow rate feeding. In the constant dissolved oxygen feeding method, the DO level was stabilized at around 20% by adjusting the glycerol feed rate. In the constant flow rate feeding method, glycerol was added at a constant flow rate of 15 g / L / h, and the DO level was naturally maintained above approximately 5%.

[0056] like Figure 4 As shown, constant-rate fed-batch glycerol feeding exhibited a faster cell growth rate throughout the fermentation cycle than constant dissolved oxygen feeding, with a more significant increase in cell wet weight. This indicates that constant-rate fed-batch glycerol feeding, by stably regulating the glycerol supply, better meets the cell growth requirements, promotes rapid biomass accumulation, and provides a solid foundation for subsequent protein expression. Therefore, constant-rate fed-batch glycerol feeding is the preferred method.

[0057] b. Effect of cell wet weight on protein yield during induction: The entire fermentation process was divided into three stages: glycerol batch culture, glycerol feeding, and methanol induction. The first two stages were consistent. During the methanol induction stage, two groups, A and B, were set up, both using pure methanol for induction at an induction temperature of 28℃ and DO controlled above 20%. Methanol induction was started for group A when the cell wet weight reached 300 g / L, and for group B when the cell wet weight reached 400 g / L. Samples were taken every 6 hours to measure wet weight and protein concentration.

[0058] like Figure 5 As shown, the total protein expression level in the supernatant of group B (induced by 400 g / L) was significantly higher than that of group A (induced by 300 g / L). This means that induction at 400 g / L can yield more bacterial biomass and higher total protein expression. Therefore, it is preferable to start methanol induction when the wet weight of the bacterial cells reaches 400 g / L.

[0059] c. Effect of basal culture medium composition on protein expression levels: BSM, D'Anjou and FM21 inorganic salt media were used as initial media, and fermentation and induction expression were carried out under the same conditions. The LECT2-b protein content in the supernatant at each time point was compared to determine the optimal basal culture medium.

[0060] like Figure 6As shown, using BSM and D'Anjou basal media resulted in relatively higher LECT2-b expression levels compared to using FM21 medium. Furthermore, D'Anjou medium is less expensive and easier to prepare than BSM, making it more suitable for large-scale production. Therefore, D'Anjou medium is preferred as the initial medium.

[0061] (3) Comparison of expression levels between KM71-LECT2-b strain and GS115-LECT2-b strain To further evaluate the effect of the host strain on LECT2-b expression, the GS115-LECT2-b strain was subjected to the same fermentation and induction expression verification based on the aforementioned optimized fermentation conditions for KM71-LECT-b (D'Anjou basal medium, constant-rate fed glycerol, 400 g / L wet weight initial induction, pH 6.0 during induction, 1.0% (v / v) anhydrous methanol, and 72 h of induction). The LECT2-b content in the supernatant of the two strains after 72 h of fermentation was compared.

[0062] like Figure 7 As shown, there was no significant difference in LECT2-b expression levels between KM71-LECT2-b and GS115-LECT2-b under the same fermentation conditions. However, during fermentation, the methanol consumption of the KM71-LECT2-b strain was significantly lower than that of the GS115-LECT2-b strain. Specifically, under the same conditions using 10L of basal medium in a 30L fermenter, the KM71-LECT2-b strain consumed approximately 2.4L of methanol, while the GS115-LECT2-b strain consumed approximately 2.7L of methanol. This indicates that using the KM71 strain to express LECT2-b protein can effectively reduce the production cost of large-scale fermentation. Therefore, considering both expression level and economic efficiency, KM71 is the preferred host strain for LECT2-b production.

[0063] (4) Determination of LECT2-b content in fermentation supernatant under optimal conditions Based on the above optimization results, the optimal fermentation conditions were determined as follows: using Pichia pastoris KM71 as the host strain, D'Anjou medium (or BSM medium) containing 4% glycerol as the basal medium, and feeding glycerol at a constant rate (feed rate 15 g / L / h). Feeding was stopped when the cell wet weight reached 400 g / L. After starvation for 1 h, the process was switched to the methanol induction stage, with an induction temperature of 28 ℃, pH 6.0, methanol feed rate of 3.7 mL / h / L, and an induction time of more than 72 h.

[0064] Under these optimal conditions, a validation experiment was conducted in a 30L fermenter. After 72 h of induction, the fermentation supernatant was collected for SDS-PAGE and Western blot analysis. The protein bands were then quantified using ImageJ software based on their grayscale values. The results are as follows: Figure 8 As shown, at a cell density of 400 g / L, the LECT2-b content in the fermentation supernatant reached a maximum of 2.2 g / L. This expression level is relatively high among the secretory expressions of antimicrobial proteins in Pichia pastoris, indicating that the fermentation process established in this invention can achieve efficient secretory expression of LECT2-b and has good potential for industrial application.

[0065] 3. Pilot-scale verification in a 2000L fermenter After optimizing the process in a 30L fermenter, pilot-scale verification was conducted in a 2000L fermenter. The operating parameters were based on those of the 30L fermenter, and the scale was increased proportionally. After 72 hours of fermentation induction, the supernatant was analyzed by SDS-PAGE. The results showed a clear LECT2-b protein band at the expected location, confirming successful expression of the LECT2-b protein in the fermentation supernatant. Figure 9 Pilot-scale results show that the KM71-LECT2-b strain can stably express LECT2-b in a 2000L fermenter, with good process reproducibility, demonstrating feasibility for industrial scale-up. This invention has been successfully validated three times on a 2000L fermenter scale, with stable process performance. These results fully demonstrate that the fermentation process established in this invention has good feasibility for industrial scale-up and is suitable for large-scale production applications.

[0066] Example 3: Purification and Activity Verification of LECT2-b 1. LECT2-b purification a. Centrifuge the fermentation broth to collect the supernatant, filter it using a 0.44μm aqueous filter membrane, and store it at 4℃ for later use.

[0067] b. Prepare binding buffer A: 3.8 mM Na2HPO4, 16.2 mM NaH2PO4, pH 7.4; prepare elution buffer B: 3.8 mM Na2HPO4, 16.2 mM NaH2PO4, 1 M NaCl, pH 7.4.

[0068] c. Using the ÄKTA protein purification system and the HiTrap SP HP ion exchange column, the LECT2-b protein was purified. Specifically, the ion exchange column was equilibrated with 500 mL of buffer A, and the filtered fermentation supernatant was loaded at a flow rate of 5 mL / min to ensure that the target protein was fully bound to the ion exchange column. After loading, the ion exchange column was equilibrated again with 500 mL of buffer A to remove unbound contaminants. Gradient elution was then performed with buffer B, and the target protein peak was collected. After elution, the ion exchange column was blocked with 20% ethanol.

[0069] d. Prepare the dialysis buffer: Add 5% glycerol to 20 mM Tris-HCl to adjust the pH to 7.4, and filter using a 0.44 μm aqueous filter membrane. Place the protein eluent collected in step c into a pre-treated dialysis bag with a molecular weight cutoff of 3500 Da, immerse it in the dialysis buffer, and dialyze at 4 ℃ for 24 h to remove salt ions and small molecule impurities.

[0070] e. The dialysis products were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the products were stained with Coomassie Brilliant Blue for 15 min, destained with boiling water until the background color was clear, and then the protein composition was observed. The results showed that the purified LECT2-b appeared as a single protein band in SDS-PAGE, indicating good purity.

[0071] 2. LECT2-b activity verification a. Take Aeromonas hydrophila strain Ah72 (isolated and preserved by our laboratory) frozen at -80℃, thaw it, inoculate it into 15ml of BHI liquid medium, incubate at 28℃ and 220 rpm for 12h, and then dilute it 10000 times for later use.

[0072] b. The LECT2-b dialysis product was filtered using a 0.22 μm syringe filter membrane. After determining the protein concentration, it was diluted to 2 mg / ml with sterile Tris-HCl (pH=7.4). 200 μl of LECT2-b solution was mixed with an equal volume of diluted Aeromonas hydrophila as the experimental group; another 200 μl of sterile Tris-HCl (pH=7.4) was mixed with an equal volume of diluted Aeromonas hydrophila culture as the control group. Both groups of samples were incubated at 28℃ for 2 h. 100 μl of the reaction solution from each group was then spread onto LB agar plates, with three replicates for each group. The plates were incubated upside down at 28℃ for 12 h, and single colonies were counted to verify the antibacterial activity.

[0073] The results are as follows Figure 10As shown, compared with the control group (Con, bacterial growth rate 100%), the growth rate of Aeromonas hydrophila Ah72 treated with LECT2-b expressed in fermenters of different sizes (30L and 2000L) was significantly reduced (****, p<0.0001). This indicates that the LECT2-b prepared by the method of this invention has a significant inhibitory effect on Aeromonas hydrophila Ah72, effectively inhibiting the growth of this pathogen, and the products under different fermentation processes maintained good biological antibacterial activity.

[0074] In summary, this invention represents the first successful application of a Pichia pastoris secretory expression system to achieve soluble secretory expression of LECT2-b. Specifically, the LECT2-b coding sequence was codon-optimized according to the codon preference of Pichia pastoris. The optimized gene was then integrated into the genome of Pichia pastoris KM71 via the pPIC9K expression vector, achieving efficient secretory expression of LECT2-b. The fermentation process was systematically optimized, and the optimal fermentation conditions were determined: using Pichia pastoris KM71 as the host strain, D'Anjou or BSM as the basal medium, and a constant-rate fed-batch glycerol feeding method (feed rate 15 g / L / h). When the cell wet weight reached 400 g / L, the process was switched to the methanol induction stage. The induction temperature was 28℃, pH 6.0, and anhydrous methanol was added at a rate of 3.7 mL / h / L to maintain the methanol concentration in the fermentation broth at approximately 1.0% (v / v). The induction time was 72 h, and the dissolved oxygen (DO) was controlled above 20%. Under these optimal conditions, the LECT2-b content in the fermentation supernatant can reach 2.2 g / L. The fermentation process of this invention can efficiently express the target protein LECT2-b with a high yield, and the product retains its natural antibacterial bioactivity, exhibiting a significant inhibitory effect on intestinal pathogens of grass carp, such as Aeromonas hydrophila. This process has been repeatedly validated on 30L and 2000L fermenter scales, demonstrating stability, good reproducibility, and good feasibility for industrial scale-up, making it suitable for large-scale production applications. This invention fills the technological gap in the secretory expression of the grass carp-derived antibacterial protein LECT2-b by Pichia pastoris, providing reliable technical support and a material basis for the promotion and application of this antibacterial protein in aquaculture disease control, and has significant industrial application value.

[0075] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A recombinant Pichia pastoris engineered strain that secretes and expresses the grass carp-derived antimicrobial protein LECT2-b, characterized in that, The engineered strain was obtained by integrating an optimized gene encoding the grass carp-derived antimicrobial protein LECT2-b into the genome of Pichia pastoris KM71 strain; the nucleotide sequence of the optimized gene is shown in SEQ ID NO.

3.

2. The recombinant Pichia pastoris engineered strain according to claim 1, characterized in that, The optimized gene was integrated into the genome of Pichia pastoris strain KM71 using the pPIC9K vector as a backbone vector.

3. The recombinant Pichia pastoris engineered strain according to claim 1, characterized in that, The method for constructing the recombinant Pichia pastoris engineered strain includes: (1) The LECT2-b coding sequence shown in SEQ ID NO.1 was optimized according to the codon preference of Pichia pastoris to obtain the optimized gene shown in SEQ ID NO.3; (2) EcoRI and NotI restriction sites were introduced at both ends of the optimized gene, while avoiding the SalI restriction site, and the gene was synthesized into the pPIC9K vector backbone to obtain the recombinant expression plasmid pPIC9K-LECT2-b. (3) Linearize the recombinant expression plasmid using SalI fast digestion enzyme; (4) Preparation of Pichia pastoris KM71 competent cells; (5) The linearized recombinant expression plasmid was transformed into Pichia pastoris KM71 competent cells by electroporation; (6) Positive transformants were obtained through G418 resistance screening; (7) Positive transformants were identified by PCR and sequenced to screen for engineered strains that highly express LECT2-b protein.

4. The application of the recombinant Pichia pastoris engineered strain according to any one of claims 1-3 in the high-yield grass carp-derived antimicrobial protein LECT2-b.

5. A method for producing grass carp-derived antimicrobial protein LECT2-b by fermentation using any one of the recombinant Pichia pastoris engineered strains according to claims 1-3, characterized in that, Includes the following steps: (1) Seed culture: The recombinant Pichia pastoris engineered strain was inoculated into seed culture medium and cultured at 25~35℃ and 200~250 rpm until the logarithmic growth phase to obtain fermentation seed liquid; (2) Fermentation tank culture: The fermentation seed liquid was inoculated into the basic culture medium at a volume ratio of 5% to 15%, and glycerol was cultured in batches at 25 to 35°C, pH 5.0 to 6.0, and dissolved oxygen ≥ 30%. (3) Glycerol replenishment: After the glycerol in the basal medium is depleted, glycerol is added at a constant rate of 10~20 g / L / h. (4) Methanol induction: Stop feeding after the wet weight of the cells reaches 200~500 g / L, adjust the temperature to 20~30℃ and the pH to 5.5~6.5, and add methanol at a flow rate of 2~5 ml / h / L for induction. The induction time is more than 72 hours. (5) Collect and purify the supernatant: After induction, centrifuge to collect the fermentation supernatant, and then purify it to obtain grass carp LECT2-b protein.

6. The method according to claim 5, characterized in that, The basic culture medium mentioned in step (2) is D'Anjou medium or BSM medium.

7. The method according to claim 5, characterized in that, In step (3), the glycerol feed rate is 15 g / L / h.

8. The method according to claim 5, characterized in that, In step (4), feeding is stopped after the wet weight of the cells reaches 400 g / L, and methanol induction begins.

9. The method according to claim 5, characterized in that, In step (4), the methanol-induced temperature was 28°C, the pH was 6.0, and the methanol flow rate was 3.7 ml / h / L.

10. The recombinant Pichia pastoris engineered strain according to any one of claims 1-3, or the method according to any one of claims 5-9, in the preparation of an antibacterial feed additive or antibacterial drug containing grass carp-derived LECT2-b protein.

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