The genes manB, manC, gmd, fcl, and futC were synthesized by General Biosystems (China). The sequences were codon-optimized for Escherichia coli expression, and the restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were cloned into the pET28a vector via the NdeI and XhoI restriction sites. E. coli BL21 (DE3) (Cat. No. D1009S) and DH5α (Cat. No. D0351) supercompetent cells were purchased from Beyotime (China). Subsequently, the recombinant plasmids were transformed into E. coli DH5α and BL21 (DE3) cells. Positive clones were selected on Luria Bertani (LB) agar plates containing 100 μg/mL kanamycin (Kana) and were verified by sequencing (Tsingke, Beijing) to obtain the recombinant engineered strains. The strains were stored at -80°C with 25% (v/v) glycerol as a cryoprotectant. The engineered strains were cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium (Cat. No. G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin.

The recombinant strains were cultured in 50 mL Erlenmeyer flasks containing 10 mL of LB medium with kanamycin at 25°C and 250 rpm. The cell concentration was measured by the optical density (OD) at 600 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). When the cell OD600 reached 0.6, the bacteria were collected by centrifugation at 4000 × g for 5 min and resuspended in 100 mL of M9 Broth (supplemented with 1 mM MgSO4 and 50 μM CaCl2) (Cat. No. A510881, Sangon Biotech, China) containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 8 g/L lactose were added to induce the production of 2′-fucosyllactose (2-FL).

The cell optical density (OD600) was measured at 600 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). Subsequently, the bacterial pellet was collected by centrifugation at 10,000 × g for 10 min and placed in an oven at 50°C for 8 h to measure the dry cell weight (DCW) of the recombinant E. coli strain.

The 2-FL engineered strain was inoculated into liquid LB medium containing 100 μg/mL kanamycin (Kana) and cultured at 25°C and 250 rpm. When the cell OD600 reached 0.6, the culture was transferred to 100 mL of M9 Broth containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM IPTG and 8 g/L lactose were added. 1 mL of the fermentation broth was collected at regular intervals. After centrifugation at 10,000 × g for 1 min, the culture supernatant was collected and stored at -20°C. The change in lactose content was analyzed using a lactose assay kit (Cat. No. JL-T1072, Jonlin). First, lactose is hydrolyzed by lactase to produce free galactose, which is further oxidized by galactose oxidase to form a product with an absorption peak at 570 nm.

Principle of the Assay Kit: Lactose is decomposed into galactose and glucose under the action of β-galactosidase. Glucose is oxidized by a specific enzyme to produce a (pink) red product that reacts with a chromogenic agent. This product has a maximum absorption peak at 510 nm. The lactose content is then obtained by correcting for the background value of free glucose.

A High-Performance Liquid Chromatography (HPLC) system (WATERS) was used to analyze the 2'-fucosyllactose produced by the engineered strain. The system was equipped with a WATERS ACQUITY UPLC BEH AMIDE column (2.1 × 100 mm, 1.7 μm) and a refractive index detector (RID). 500 μL of the fermentation sample (broth) was mixed with 500 μL of acetonitrile. Then, after centrifugation at 12,000 × g for 5 minutes, the supernatant was filtered through a 0.22 μm filter membrane. The mobile phase was A: 80% acetonitrile and 20% ammonia water (0.1%); B: 30% acetonitrile and 70% ammonia water (0.1%). The samples were detected at a flow rate of 0.3 mL/min and a temperature of 45°C. According to the program gradient, from 0-7 min, mobile phase A decreased from 90% to 60%, and from 7-10 min, it was restored to 90%. From 0-7 min, mobile phase B increased from 10% to 40%, and from 7-10 min, it decreased to 10%.

The 2-FL engineered strain was inoculated into liquid LB medium containing 100 μg/mL kanamycin (Kana) and cultured at 25°C and 250 rpm. When the cell OD600 reached 0.6, the cells were centrifuged and resuspended in 100 mL of M9 Broth containing 20 g/L glycerol and 5 g/L yeast extract. When the cell OD600 reached 0.6, 0.5 mM IPTG and 8 g/L lactose were added. 1 mL of the fermentation broth was collected at regular intervals. 1 mL of the fermentation broth was collected. After centrifugation at 10,000 × g for 1 min, the culture supernatant was collected and stored at -20°C. The 2-FL content in the fermentation broth of the engineered strain was analyzed using a 2′-Fucosyllactose (2′-FL) Assay Kit (Cat. No. 680112, Synome) according to the manufacturer's instructions.

The open reading frame (ORF) of TRYP (UniProt: P00761) was codon-optimized to comply with the RFC#10 standard, synthesized, and inserted into the NdeI and XhoI digested pET-28a plasmid to generate the pET-28a-TRYP recombinant vector. The vector was transformed into the E. coli BL21 (DE3) (Cat. No. D1009S, Beyotime, China) strain. Positive clones were selected on Luria Bertani (LB) agar plates containing 100 μg/mL kanamycin (Kana) and were verified by sequencing (Tsingke, Beijing) to obtain the recombinant engineered strain. The strain was stored at -80°C with 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium supplemented with 100 μg/mL kanamycin.

Expression and Collection of Inclusion Bodies: BL21-pET-28a-TRYP was inoculated into a 250 mL flask containing 50 mL of LB medium with 100 μg/mL kanamycin and cultured in a shaker at 37°C and 150 rpm. When the OD600 reached 0.6, 0.5 mM IPTG was added, and the culture temperature was adjusted to 16°C for a 20 h induction. Subsequently, the bacterial pellet was collected by centrifugation (10,000 × g, 5 min) and resuspended in pre-chilled PBS (pH = 7.5). Sonication was performed using an ultrasonic cell disruptor (Jingxin, Shanghai, China) (70 W, 3 s interval, 1 s pulse, 20 min). Subsequently, the inclusion body pellet was collected by centrifugation at 10,000 × g for 30 min at 4°C.

Inclusion Body Refolding: The pellet was washed with PBS containing 2 M urea (Cat. No. A68476, Innochem, China) and incubated for 1 h at 25°C and 150 rpm. Subsequently, it was centrifuged at 10,000 × g for 30 min at 4°C. 10 mL of denaturation solution (20 mM Tris, 8 M urea, 20 mM DTT, pH 8.5) was added per gram of inclusion body pellet, and it was incubated for 4 hours at 25°C on a destaining shaker. The supernatant was collected after centrifugation at 12,000 rpm for 20 min. The supernatant was added to the refolding buffer (20 mM Tris, 1 mM cystine, and 3 mM cysteine, 1 M urea; pH = 9) at a 1:40 dilution ratio and stirred overnight at 4°C.

Zymogen Activation: TRYP was further concentrated using a 15 mL ultrafiltration tube (10 kDa, Millipore) and resuspended in Tris buffer (40 mM Tris, 0.1 M NaCl, 10 mM CaCl2, pH 8.0). After activation for 1 hour at 25°C, the protein concentration was determined using a Bradford assay kit (Cat. No. P0006, Beyotime, China). It was aliquoted and stored frozen at -80°C.

Explanation of Zymogen Activation Mechanism: In this experimental protocol, the activation of trypsinogen is based on a self-catalytic conformational transition mechanism: the trypsinogen, concentrated by ultrafiltration, is activated in a specific buffer system (40 mM Tris, 0.1 M NaCl, 10 mM CaCl₂, pH 8.0) at 25°C through the self-cleavage of the N-terminal propeptide sequence.

Specifically, Ca²⁺ in the buffer stabilizes the spatial conformation of trypsinogen, promoting the exposure of its catalytic active site; the weakly alkaline environment (pH 8.0) facilitates intermolecular interactions among trypsinogen molecules, initiating the spontaneous cleavage of the propeptide in a small number of molecules to generate active trypsin. The generated trypsin, through a positive feedback loop, further efficiently cleaves the propeptides of the remaining trypsinogen, ultimately completing the conversion from the inactive zymogen to the active trypsin. This process does not require an external activator (such as enterokinase) and achieves self-activation of the zymogen by optimizing the in vitro environment (ionic strength, temperature, pH).

What are inclusion bodies: Inclusion bodies are insoluble, inactive protein aggregates that form in the cytoplasm or periplasmic space when cells express recombinant proteins, due to overly rapid protein synthesis, incorrect folding, or the lack of necessary folding-assisting factors. They are typically granular in appearance and require denaturation and refolding treatments to restore protein activity.

The trypsin activity of TRYP was determined using a Trypsin (TRY) Activity Assay Kit (Cat. No. AKPR005M, boxbio) according to the instructions. Trypsin is capable of catalyzing the hydrolysis of the ester bond in N-Benzoyl-L-Arginine-Ethylester (BAEE) to produce N-Benzoyl-L-Arginine (BA), and the product has a characteristic absorption peak at 253 nm. After incubating for 5 min, the absorbance at 253 nm was measured using a microplate reader (FlexStation 3, Molecular Devices, USA). One unit of enzyme activity is defined as the amount of enzyme that causes an increase in absorbance of 0.0005 at 253 nm per minute per mg of protein in a 1 mL reaction system at 25°C.

Official website link and product documentation for the kit:https://www.boxbio.cn/product/772.html?goodsno=AKPR005M

Western Blot Analysis of TRYP Expression

The engineered bacterial strain overexpressing TRYP was inoculated into 50 mL of Luria-Bertani (LB) medium supplemented with ampicillin and cultured overnight. The following day, the bacterial culture was collected by centrifugation at 10,000 × g for 1 minute. The resulting cell pellet was resuspended in PBS and subsequently lysed by sonication (150 W, with cycles of 1 second on and 3 seconds off, for a total of 20 minutes). The lysed cell suspension was then transferred to a new centrifuge tube and designated as the whole-cell lysate sample.

The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 ratio and denatured in a boiling water bath for 15 minutes. Proteins were then separated by 12% SDS-PAGE at 120 V. Subsequently, the proteins were transferred from the SDS-PAGE gel to a polyvinylidene fluoride (PVDF) membrane under ice-cold conditions. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature.

Given that the pET vector incorporates a His-tag, the membrane was incubated overnight at 4°C with a mouse anti-His-tag primary antibody (1:1000 dilution, Cat# AH367, Beyotime). After incubation, the membrane was washed three times with TBST for 10 minutes each. It was then incubated for 1 hour at room temperature with a Horseradish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat# A0216, Beyotime).

Following three final 10-minute washes with TBST, the chemiluminescence signal was developed. ECL solutions A and B were mixed at a 1:1 ratio immediately before use. The washed PVDF membrane was briefly placed on absorbent paper to remove excess surface liquid, then laid on the tray of the imaging system. The prepared ECL substrate was applied to completely cover the membrane. After a 1-minute reaction, excess substrate was wicked away, and the membrane was placed into the chemiluminescence imaging system to capture the signal according to a preset program.

The open reading frame (ORF) of LacZ was codon-optimized to conform to the RFC#10 standard, synthesized, and subsequently inserted into a pET-28a plasmid that had been digested with NdeI and XhoI, thereby generating the recombinant vector pET-28a-LacZ. This vector was then transformed into the E. coli BL21(DE3) strain (Cat# D1009S, Beyotime, China). Positive clones were selected on Luria-Bertani (LB) plates supplemented with 100 μg/mL kanamycin (Kan) and validated by sequencing (Tsingke, Beijing) to obtain the final recombinant engineered strain. The strain was preserved at -80°C using 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm. Inoculation and expansion cultures were performed in liquid LB medium containing 100 μg/mL Kan.

The engineered bacterial strain overexpressing LacZ was inoculated into 50 mL of LB medium supplemented with ampicillin and cultured overnight. The following day, the bacterial culture was harvested by centrifugation at 10,000 × g for 1 minute. The cell pellet was resuspended in PBS and lysed by sonication (150 W, operating in cycles of 1 second on and 3 seconds off, for a total duration of 20 minutes). The resulting lysate was transferred to a new centrifuge tube to serve as the whole-cell lysate sample.

The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 (v/v) ratio and denatured in a boiling water bath for 15 minutes. The proteins were separated by 12% SDS-PAGE at 120 V. Subsequently, the proteins were transferred from the SDS-PAGE gel to a polyvinylidene fluoride (PVDF) membrane under ice-cold conditions. The membrane was blocked for 1 hour at room temperature with 5% non-fat milk prepared in TBST. As the pET vector contains a His-tag, the membrane was incubated overnight at 4°C with a mouse anti-His-tag primary antibody (1:1000 dilution, Cat# AH367, Beyotime). The membrane was then washed three times with TBST, for 10 minutes each. Following this, it was incubated for 1 hour at room temperature with a Horseradish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat# A0216, Beyotime).

After three final 10-minute washes with TBST, ECL solutions A and B were mixed at a 1:1 ratio for immediate use. The washed PVDF membrane was placed on absorbent paper to briefly remove excess surface liquid, then positioned on the tray of a chemiluminescence imaging system. The prepared ECL substrate was applied to ensure the membrane was completely submerged. After a 1-minute reaction, excess liquid was wicked away from the top, and the membrane was placed into the imaging system to initiate the chemiluminescence reaction according to a preset program.

Purified LacZ enzyme was added to a 1 mL reaction system containing an initial lactose concentration of 10 mM, achieving a final enzyme concentration of 0.1 U/mL. The reaction was incubated at an optimal temperature, and samples were collected at 0, 2, 4, 6, and 8 hours. The production of glucose, which serves as an indirect measure of lactose consumption, was quantified using the Abcam Glucose Assay Kit (Cat# ab272532).

The specific procedure was as follows: 5 μL of either the glucose standard or the test sample was added to a 1.5 mL centrifuge tube. To this, 500 μL of the detection reagent was added, and the solution was mixed by vortexing. The tube was then tightly capped and precisely heated in a boiling water bath for 8 minutes, followed by cooling in an ice-water bath for 4 minutes. A 200 μL aliquot of the reaction solution was transferred to a 96-well plate. The absorbance was measured at a wavelength of 630 nm using a microplate reader. The glucose concentration was calculated based on the standard curve, which was then used to determine the amount of lactose degraded.

GraphPad Prism software was used for data analysis and plotting. Data are presented as mean ± standard deviation (SD). For comparisons of multiple groups, one-way analysis of variance (One-way ANOVA) followed by Tukey's post hoc test was used for difference analysis. For comparisons between two groups, a Student's t-test was used for analysis. A p-value of less than 0.05 was considered statistically significant.