Efficient Expression of Small Molecule Bioactive Peptides in Bacillus licheniformis

Efficient Expression of Small Molecule Bioactive Peptides in Bacillus licheniformis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient Expression of Small Molecule Bioactive Peptides in Bacillus licheniformis Hanchao Zhang, Lanying Shao, Qinghua Feng, Qingping Guo, Haikuan Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8706144/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Stable expression of γ-Glutamyltranspeptidase (GGT)-BPC157 and mScarlet-BPC157 in Bacillus licheniformis strain 2709 by chromosomal integration. Fermentation conditions were optimized using single-factor and orthogonal experiments to maximize yield. Under optimal conditions (2% inoculum, 40g/L soybean peptone, and 80g/L glucose), target protein titers increased threefold compared to the basal medium. The fusion proteins were purified by fractional ammonium sulfate precipitation; optimal saturations were 50% and 60%, respectively. Biological activity was assessed in a rat model of ethanol-induced acute gastric ulcer. Both proteins showed gastroprotective and therapeutic activity, with ulcer inhibition rates ranging from 75.3% to 82.9% protective regime and 80.4% to 84.6% therapeutic regime. Histological analysis showed that the treatment reduced mucosal inflammation and stimulated glandular repair. ELISA showed significant downregulation of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in gastric tissue. These results suggest a biotechnological production strategy for BPC 157 and applications for gastric ulcer treatment. Bacillus licheniformis BPC 157 Chromosomal integration Fusion protein Fermentation optimization Gastric ulcer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Bioactive peptides can play a variety of physiological roles, such as antioxidation, antibacterial, and immunomodulation[ 1 , 2 ], so they have become a key research object in the field of drug research and development and functional food. At the same time, it can also be used as a growth additive[ 3 ] and a feed additive, which can help improve nutrient absorption efficiency and maintain intestinal health[ 4 , 5 ]. Compared with macromolecular proteins, small peptides with a molecular weight of 1–10 kDa have higher bioavailability and better stability, and can achieve therapeutic effects with lower dosage. Among these small peptides, BPC 157 (body protection compound − 157) has attracted a lot of attention. It was first isolated from gastric juice. Although its molecular weight became smaller, it still retained the cytoprotective function of the parent protein[ 6 ]. It is not afraid of hydrolysis in the stomach, but also can promote tissue repair of multiple organs from the gastrointestinal tract to soft tissue[ 7 ]. In addition, BPC 157 can also regulate inflammatory reaction and promote angiogenesis, which is realized by interacting with the nitric oxide (NO) system[ 8 ]. The clinical application prospect of BPC 157 is very good, but its industrial production still faces many bottlenecks. The chemical solid-state synthesis method has a high cost, but it also uses harmful solvents, and it is difficult to expand the production scale[ 9 ]. In contrast, heterologous biosynthesis is more sustainable and cheaper. However, the recombinant expression of short peptides often encounters the problems of intracellular protease degradation and low downstream purification efficiency[ 10 ]. Fusion of the target peptide with a stable carrier or functional label can improve the stability of the peptide and make the recovery process simpler. Choosing the right host strain is the key to realizing efficient production. B. licheniformis is classified as a GRAS (generally considered safe) strain, which has two characteristics: it can efficiently secrete protein out of cells, and it can also grow by using cheap carbon sources[ 11 , 12 ]. With the development of systems biology, B. licheniformis has become a multifunctional cell factory for producing enzymes and peptides[ 13 ]. The expression system based on a plasmid can achieve high yield, but it often brings a metabolic burden to the host, and there are also problems of separation instability. In addition, antibiotics cannot be used in food-grade products[ 14 ]. Therefore, integrating an expression cassette into a chromosome has obvious advantages, which can ensure genetic stability without screening pressure. If we combine the strong endogenous signal peptide and optimize the promoter strength through metabolic engineering[ 15 ], the chromosome-encoded construct can achieve a reliable secretion level suitable for industrial fermentation. This paper introduces a simple biological process for producing BPC 157 in B. licheniformis. The researchers obtained stable strains through chromosome integration and fusion gene constructs, and also optimized fermentation parameters to improve the yield. In addition, the team developed an efficient purification process and evaluated the biological activity of the recombinant peptide through gastric mucosal protection experiments in vitro. These findings provide a method for large-scale biological production of BPC 157. Materials and methods Strains, plasmids, and reagents The following strains were used in this study: B. licheniformis 2709, maintained in our laboratory, served as the host for expressing fusion proteins. Escherichia coli JM109 and Escherichia coli EC135pM. Bam was obtained from the Institute of Microbiology, Chinese Academy of Sciences (IMCAS, Beijing, China) for plasmid construction and propagation. The shuttle vector pKSVT was supplied by Hubei University (Wuhan, China), while plasmid pTU-apr is part of our laboratory stock. PrimeSTAR Max DNA Polymerase and the restriction endonucleases SmaI, SacI, SpeI, and NotI were acquired from Takara Biomedical Technology (Beijing, China). Additionally, antibiotics such as kanamycin and spectinomycin hydrochloride were sourced from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade and readily available. DNA manipulation and strain construction The coding sequence for BPC 157 was obtained from the Usp-BPC157 fusion entry in GenBank. Given that the choice of fusion partner significantly influences the secretion efficiency of recombinant peptides [ 16 ], we selected GGT subunits (21–25 kDa and 41–65 kDa) to enhance the effective mass of the peptide [ 17 ]. We constructed a fusion protein that directly links BPC 157 to the GGT subunit, incorporating an N-terminal 6×His tag. Design primers for amplifying the sequence encoding BPC 157 and subsequent validation(Table S1 ). For the reporter assay, we fused the coding sequence of the red fluorescent protein mScarlet to BPC 157, also with an N-terminal 6×His tag. All target sequences were synthesized by BGI (Beijing, China). The pKSVT-GGT-BPC157 vector was created by combining the GGT insert with the pKSVT shuttle vector. The mScarlet-BPC157 fusion fragment was amplified from the plasmid template using primers BR-F and BR-R. Homologous arms flanking the GGT gene were amplified from B. licheniformis 2709 genomic DNA with primer pairs GGT-UF/GGT-UR and GGT-DF/GGT-DR (Fig. S1 c). The pKSVT vector underwent linearization through double digestion with SpeI and NotI (Fig. S1 a), followed by the ligation of the homologous arms into the vector (Fig. S1 b). The products were transformed into E. coli JM109. Verified plasmids were extracted and subsequently transformed into the methylation-proficient strain E. coli EC135pM. Bam. Transformants were selected on plates containing kanamycin and spectinomycin, and plasmid methylation was initiated by the addition of L -arabinose. The methylated plasmids were isolated and electroporated into competent B.licheniformis 2709 cells, with transformants selected on kanamycin plates. Single-crossover integration was achieved by culturing at 42°C, followed by colony PCR (Fig. S2a). After incubation at 37°C in antibiotic-free medium, double-crossover events and plasmid backbone excision were conducted. The final integrated GGT- BPC157 sequence was verified through DNA sequencing (Fig. S2b). To create the recombinant strain of mScarlet-BPC157 using the integration vector pTU-apr, the mScarlet-BPC157 fusion gene is cloned into pTU-apr, resulting in the recombinant plasmid pTU-apr-mScarlet-BPC157 (Fig. S3a). Subsequent steps involve methylation, electroporation, and allelic exchange, following the same procedures used for constructing the GGT-BPC157 strain (Fig. S3b). The final recombinant strain confirms the integration of mScarlet-BPC157 at the genomic aprE locus (Fig. S3c). Determination of the growth curve of recombinant strains Recombinant strains were revived from glycerol stocks by streaking onto non-selective LB plates. After isolation of single colonies, they were cultured in 5 mL of LB broth at 37°C with shaking at 220 rpm for 12 h. Subsequently, 1 mL of the starter culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh LB medium. The optical density at 600 nm (OD 600 ) was measured every 2 h over a 24 h period to monitor cell growth. The obtained absorbance values were utilized to generate a growth curve[ 18 ]. SDS-PAGE detection of fusion proteins SDS-PAGE detection of fusion proteins Recombinant strains were revived from glycerol stocks by streaking onto antibiotic-free LB agar and incubating inverted at 37°C overnight. Single colonies were inoculated into liquid LB and shaken at 220 rpm[ 19 ], 37°C for 12–14 h. From this overnight culture, 1 mL was transferred into a 250 mL flask containing 50 mL LB and incubated until the optical density at 600 nm (OD 600 ) reached 0.8–1.0. This culture was then used as inoculum at (2% v/v) into the production medium and incubated under the appropriate conditions until protein expression reached its maximum. Cells were harvested by centrifugation at 10,000× g for 20 min at 4°C, and the resulting supernatant was collected for downstream processing. Protein samples were analyzed by SDS-PAGE following a three-step procedure. First, 20 µL of the supernatant was mixed with 5 µL of 5× loading buffer and denatured at 100°C for 5 min [ 20 ]. Electrophoresis was carried out at 80 V through the stacking gel and then at 120 V through the resolving gel until the bromophenol blue dye front reached the bottom. Gels were washed with ultrapure water and stained using a rapid microwave-assisted Coomassie protocol: the gel was briefly boiled in water in the microwave for 15–30 s, cooled in Coomassie Brilliant Blue solution with a short heating step, and then stained for 30 min. Finally, gels were destained in ultrapure water, replacing the water every 2 h until the background was clear. Determination of mScarlet-BPC157 fusion protein expression levels Shake-flask fermentations were carried out in 500 mL flasks using the sequence-verified recombinant strain B. licheniformis (BL-apr-mScarlet-BPC157); a parental strain lacking the target genes served as the negative control. All fermentations were performed in biological triplicate. Samples were withdrawn every 12 h, and culture supernatants were collected and diluted as required. To quantify target protein expression, 200 µL of each supernatant was transferred to black, opaque 96-well plates (three technical replicates per biological sample). Fluorescence was recorded on a multifunctional microplate reader with excitation and emission wavelengths of 569 nm and 594 nm, respectively [ 21 ]. Western blot verification of the fusion protein Western blot was carried out on precast denaturing SDS-PAGE gels (Yisheng Biotechnology, Shanghai, China). Before use, the transfer buffer was chilled to -20°C. Proteins were then wet-transferred to a nitrocellulose (NC) membrane at a constant 200 mA for 50 min. Following the transfer, the membrane was washed on an orbital shaker with 1× TBST for 5, 10, and 15 min sequentially, then blocked with 5% skim milk at room temperature (22–25°C) for 60 min. After three additional TBST washes, the membrane was incubated with HRP-conjugated mouse anti-His tag antibody, washed again, and the protein bands were detected using ECL working solution and a chemiluminescence imaging system. Optimization of shake flask fermentation conditions for Bacillus licheniformis We used the fluorescence intensity of the mScarlet fusion protein as a quantitative readout of recombinant protein production in BL-apr-mScarlet-BPC157 at the reported peak expression time [ 22 ]. Fluorescence measurements were performed after centrifugation and appropriate dilution to control for variations in medium composition and fermentation conditions. Initial screening experiments examined single-factor effects of carbon and nitrogen sources. On an equimolar basis, soluble starch, glucose, lactose, and sucrose each replaced the basal carbon source(cornmeal); optimal carbon source concentration was then evaluated across the range 48 to 96 g/L. For organic nitrogen sources, soybean peptone, soybean powder, soybean meal powder, yeast extract, and peanut meal powder were assessed at a constant total nitrogen content, with supplementation rates varied from 20 to 80 g/L[ 23 ]. Inoculum size was subsequently examined between 1% and 4% v/v using the identified optimal basal medium. Following single-factor screening, three influential variables—carbon source concentration, nitrogen source concentration, and inoculum size—were optimized in combination using an L 9 (3 3 ) orthogonal array design. Each experimental condition was performed in triplicate. The original medium served as the negative control, and fluorescence intensity was used as the primary response variable. Ammonium sulfate staged precipitation The strain was cultivated in the optimized medium, and fermentation-broth samples were harvested at peak expression. Initial SDS-PAGE analysis was performed, after which the culture was centrifuged at 10,000× g for 30 min, and the target protein was enriched by ammonium sulfate fractionation. The clarified supernatant was aliquoted into 20 mL portions, and saturated ammonium sulfate solution was added incrementally to achieve successive 10% saturation steps. Each increment was mixed by magnetic stirring for 5–10 min and then held at 4°C for 1–2 hours to allow precipitation. Precipitated material was collected by “salting out” followed by centrifugation at 10,000 RPM for 20 min; pellets were recovered and reconstituted in phosphate buffer. Both the supernatant and the reconstituted precipitate were transferred separately into dialysis bags and dialyzed at 4°C against 0.01 M PBS. To remove residual ammonium sulfate, the dialysis buffer was replaced 6–8 times. To identify the optimal ammonium sulfate saturation range for primary recovery of the target protein, we analyzed both the dialyzed supernatant and pellet by SDS-PAGE. Remaining fermentation broth within the determined ammonium sulfate saturation range was processed to further concentrate the target protein [ 24 ]. Protein concentrations of the resulting fractions were measured by the BCA assay, and samples were stored at 4°C for subsequent studies on the prevention and treatment of gastric ulcers. Establishment of the application model of fusion proteins in gastrointestinal inflammation A total of 90 healthy male Wistar rats (200 ± 20 g) were selected and acclimated for one week at room temperature (22 ± 2 ℃). After acclimation, rectal temperature was measured to establish baseline values. Male Wistar rats ( n = 90) were acclimated for one week at 22 ± 2°C, and baseline rectal temperatures were recorded. Two fusion proteins (GGT-BPC157 and mScarlet-BPC157) were prepared in 0.01 M PBS at 70 mg/mL, and standard BPC 157 was prepared at 10 ng/kg. Animals were assigned to protective and therapeutic experimental paradigms. For the protective model, 30 rats were randomly allocated into five groups ( n = 6 per group): Normal, Model, GGT-BPC157, mScarlet-BPC157, and Positive Control. Treatments were administered once daily by oral gavage (5 mL/kg) for 7 days; the Normal and Model groups received an equivalent volume of PBS. After the final dose, rats were fasted for 24 h. Gastric injury was then induced in all groups except the Normal control by intragastric administration of 95% ethanol (5 mL/kg). Animals were euthanized 4 h after gastric injury induction.In the therapeutic model, gastric injury was induced before treatment allocation[ 25 , 26 ], after which rats were administered the treatment once daily for 7 days. Animals underwent a 24-hour fast before euthanasia. Measurements of gastric mucosal lesions were quantified by measuring the ulcer’s maximal longitudinal diameter ( d 1 ) and maximal transverse width ( d 2 ) with a vernier caliper; ulcer area was calculated as S = π × d 1 /2 × d 2 /2. Ulcer inhibition rate was then determined. Concentrations of TNF-α, IL-1β, and IL-6 in gastric tissue were measured by ELISA. Gastric specimens were processed, sectioned, and stained with hematoxylin-eosin (HE) for histological evaluation. Data were plotted and analyzed using GraphPad Prism 10 (GraphPad Software, Boston, MA, USA). Statistical comparisons employed one-way analysis of variance followed by t-tests. Significance thresholds were set as follows: 0.05 < P < 0.01, 0.01 < P < 0.001, P < 0.001, and P < 0.0001. Biochemical assays were performed by Tianjin Hongke Biotechnology Co., Ltd. (Tianjin, China). Results Determination of the growth curve of recombinant strains Growth curves illustrate bacterial proliferation under defined culture conditions. The recombinant strains B. licheniformis (BL2709-GGT-BPC157) and B. licheniformis (BL2709-mScarlet-BPC157) exhibit growth profiles that closely parallel the parent strain B. licheniformis 2709 (Fig. 1 ). All three strains show low OD 600 values and slow growth during the initial culture period. In that early phase, the wild-type B. licheniformis 2709 increases in optical density slightly faster than the two recombinant strains. Approximately 16 h after inoculation, all strains enter the stationary phase. The OD 600 maxima of the two recombinants are comparable to one another but are marginally lower than the wild type. Thus, although the overall growth patterns remain essentially unchanged, the recombinant strains reach similar exponential-phase growth rates as the wild type while attaining a modestly lower final cell density in the stationary phase, likely reflecting the metabolic burden of heterologous gene expression. Because the fundamental growth dynamics are preserved, inocula for fermentation and optimization can be taken from the late logarithmic to early stationary phase. Fermentation of recombinant strains Single colonies of the successfully constructed strains were inoculated into 5 mL LB and grown overnight at 37°C with shaking. Cultures were then added 1:50 into 50 mL of fresh LB (2% inoculum). When the culture reached OD 600 of 0.6–0.8, cells were transferred into the fermentation medium at a 2% inoculum [ 27 ]. Previous work indicates that GGT activity peaks after 72 h of fermentation. Since the BPC 157 is fused to a subunit of GGT and has a relatively low molecular weight, the secretion level of the fusion protein was indirectly assessed by measuring GGT activity. Correctly sequenced strains and their parental controls were cultivated in the fermentation medium, and samples were collected every 12 h throughout the fermentation cycle to monitor extracellular fluorescence. Fluorescence increased progressively, reaching approximately 60,000 AU at 72 h, while the parental negative control showed no detectable signal (Fig. 2 ). These results indicate that mScarlet-BPC157 was successfully expressed in B. licheniformis and secreted into the medium. Because the gastric pentadecapeptide is fused to the red fluorescent protein, fluorescence intensity serves as a surrogate measure of the peptide’s expression and secretion. Using this proxy, the recombinant strain’s protein expression peaked at 72 hours of fermentation. SDS-PAGE validation of recombinant strains Shake-flask fermentations of the recombinant strains were carried out, and samples were collected at the secretion peak (Fig. 2 ). Following centrifugation (13,000× g, 2 min), supernatants were recovered for analysis. Supernatants from the parental strain B. licheniformis 2709 grown under identical conditions served as the negative control. Protein expression was assessed by SDS-PAGE. The theoretical molecular weight of GGT-BPC157 is 23 kDa, yet the predominant band migrated near 25 kDa (Fig. 3 a); no corresponding bands appeared in the negative control, and the upward shift is consistent with post-translational modification. In contrast, mScarlet-BPC157 produced a single band at the expected molecular weight (Fig. 3 b). Collectively, these data indicate that both recombinant B. licheniformis strains secreted their respective fusion proteins. Western blot identification of recombinant fusion proteins The expression of the recombinant fusion proteins was confirmed by Western blot targeting the C-terminal 6×His tag. Immunodetection was performed using an HRP-conjugated mouse anti-His tag monoclonal antibody. The fermentation supernatant of the parental B. licheniformis 2709 strain was used as a negative control (Fig. 4 a). After electrophoretic separation and transfer to NC membranes, the antibody revealed distinct immunoreactive bands of GGT-BPC157 and mScarlet-BPC157. The absence of specific signals in the negative control (Fig. 4 b) confirmed the specificity of the detection. This shows that both His-tag fusion proteins were successfully expressed. The influence of the types and addition amounts of carbon sources on the fermentation of recombinant Bacillus licheniformis Carbon source identity and concentration strongly influence recombinant bacterial fermentation because these substrates supply both the structural carbon and the energy required for growth and metabolism [ 28 ]. To quantify this effect, we examined how alternative carbon sources affected fermentation of B. licheniformis , using the mScarlet fluorescence intensity as a proxy for recombinant protein expression. We selected corn flour (64 g/L) as the reference carbon source and compared it to soluble starch, glucose, lactose, and sucrose, each supplied at an equivalent carbon content. For each carbon source, experiments were run in triplicate. RFP fluorescence, measured from the culture supernatant, served as the readout for secretion efficiency and expression level. Carbon source type had a pronounced effect on protein expression (Fig. S4a). Glucose yielded the highest fluorescence, followed by sucrose and lactose; soluble starch and corn flour produced the lowest signals. The difference in fluorescence between glucose and corn flour was statistically significant, indicating that B. licheniformis preferentially exploits mono- and disaccharides over polysaccharides for recombinant protein production. Based on these results, glucose was selected as the carbon source for subsequent experiments. To optimize the addition of glucose, we set four concentration gradients (48, 64, 80, and 96 g/L) were evaluated in triplicate. Results showed that fluorescence intensity increases slowly from 48 to 80 g/L. A peak was observed at 96 g/L, but the expression level at 80 g/L was slightly lower than that at 64 g/L.These results show that increasing glucose concentration can increase protein expression even if it is below the threshold. We chose 64, 80, and 96 g/L as carbon source levels for orthogonal experiments (Fig. S4b) The influence of the types and addition amounts of nitrogen sources on the fermentation of recombinant Bacillus licheniformis Nitrogen source type and concentration strongly affect fermentation performance and recombinant protein expression in bacteria because they determine the availability of nitrogenous precursors and thereby influence core metabolic activity [ 29 ]. To quantify these effects, we evaluated the impact of several complex nitrogen sources on the fermentation of recombinant B. licheniformis while keeping all other culture conditions constant. Glucose was fixed at 64 g/L as the carbon source. Using soybean meal powder as a control, four composite nitrogen sources (all with a concentration of 40 g/L) were tested: soybean peptone, soybean powder, yeast extract, and peanut meal powder. We measured expression of a red fluorescent protein reporter in the recombinant strains to compare how each nitrogen source modulated protein production. The type of nitrogen source has an obvious influence on the expression of the reporter gene (Figure S5a): the fluorescence intensity of the soybean peptone group is the highest, which indicates that it is most suitable to support the synthesis of recombinant protein under experimental conditions. The expression level of the soybean powder group was the second, and the fluorescence intensity of the yeast extract and peanut meal powder group was much lower. This result shows that even if the nitrogen content is the same, the source and formula of the compound nitrogen source are different, which will have different effects on the metabolic state of bacteria and the yield of recombinant protein. Based on the results, soybean peptone was chosen as the best addition amount. Four concentration levels were set at 20, 40, 60, and 80 g/L. From 20 g/L to 40 g/L, fluorescence intensity increased gradually, peaked at 40 g/L, but decreased at 60 and 80 g/L. High nitrogen concentrations may hinder growth and protein expression (Fig. S5b). Soybean peptone in the appropriate amount can increase protein expression of recombinant bacteria. In orthogonal experiments, three supplementation levels (20 g/L, 40 g/L, 60 g/L) were chosen as nitrogen source levels. The influence of inoculation size on the fermentation of recombinant Bacillus licheniformis Inoculation size directly affects the growth and metabolic efficiency of bacteria: insufficient inoculation size will prolong fermentation time and reduce yield[ 30 ], while excessive inoculation size may inhibit production efficiency or metabolic activity. In order to quantify its influence on the synthesis of recombinant protein, the researchers compared four inoculation sizes of 1%, 2%, 3% and 4%(v/v) and detected the expression of fluorescent reporter protein. The fluorescence intensity increased with the increase of inoculation size, and the fluorescence of 1% inoculation group was significantly lower than that of other groups because of its low initial cell density and long delay period. The fluorescence intensity of the 4% inoculation group reached the peak (Figure S6). These results indicate that increasing the inoculation density can promote the growth of bacteria and increase the expression of target protein. Therefore, the orthogonal experiment selected 2%, 3% and 4% as the inoculation level. Orthogonal experiment Single-factor optimization identified three fermentation variables— inoculum size (A), soybean peptone concentration (B), and glucose concentration (C)—each tested at three levels. We then organized an orthogonal experimental design using these factors and employed fluorescent protein expression intensity as the response variable. Range analysis of the resulting data ranked the factors by influence on protein expression as glucose (C) > soybean peptone (B) > inoculation volume (A) (Table S2). That analysis also indicated that Experiment 2 produced the highest aggregated expression level. Further Analysis of the mean values ( K ), determined the optimal factor combination as A1B2C2, corresponding to an inoculum size of 2% (v/v), 40 g/L soybean peptone, and 80 g/L glucose (Fig. S7a). Fermentation carried out with this optimized medium yielded a fluorescence intensity roughly threefold greater than that obtained with the original medium, demonstrating that the optimized conditions substantially enhanced the recombinant strain’s protein expression capacity (Fig. S7b). Ammonium sulfate fractionation and precipitation of fusion proteins The recombinant strain was cultivated in shake flasks using the optimized medium, and the culture broth was harvested at the time of maximal protein expression. Ammonium sulfate precipitation was carried out as described to precipitate GGT-BPC157 and mScarlet-BPC157. After precipitation, SDS-PAGE analysis was performed on both the supernatant and the precipitate. GGT-BPC157 began to precipitate at 40% ammonium sulfate saturation, but the resulting precipitate contained noticeable impurities. Raising the saturation to 50% caused most of the fusion protein to precipitate while reducing contaminating bands (Fig. S8a, b). In contrast, mScarlet-BPC157 did not begin to precipitate until 60% saturation; at this point, the target protein precipitated selectively, with relatively low levels of impurities. Increasing the saturation further to 90% precipitated most proteins present in the fermentation broth. Consequently, 50% and 60% saturation levels were identified as optimal for the purification of GGT-BPC157 and mScarlet-BPC157, respectively (Fig. S8c, d). The ammonium sulfate precipitation on large volumes of broth (optimal saturation) was resuspended with 0.02 M PBS and re-dissolved in a boiling EDTA (3.5 kDa) dialysis bag, 4°C, 0.02 M PBS, 24 hours. The dialysate was replaced until no flocculent precipitation was observed with 1% BaCl 2 . The fusion protein was measured using the BCA kit. The effect of fusion protein on the gastric morphology of ethanol-induced acute gastric ulcer in a rat model Concentration of purified fusion protein measured with BCA kit (70 µg/mL) in 0.01 M PBS buffer [ 31 ], and administered according to the method. Gastric ulcer models were induced simultaneously in the protection and treatment groups. After modelling gastric mucosal morphology of rats in each group was assessed. Figure results for the protection cohort are presented in (Fig. 5 a). The gastric mucosa of the normal control group was intact and unremarkable, with no signs of congestion, edema, or structural damage. In contrast, the model group exhibited extensive injury, including cord-like or patchy hemorrhages, blackish-brown ulcers, and pronounced gastric bleeding. The BPC 157 positive-control group showed a clear therapeutic response. Both the GGT-BPC157 and mScarlet-BPC157 groups demonstrated only mild improvement, with sporadic ulcer foci remaining. Results for the treatment cohort are shown in (Fig. 5 b). The normal group retained an intact, undamaged gastric inner wall, whereas the model group continued to display patchy hemorrhage and mucosal erosion. After treatment, gastric tissue in the experimental groups closely resembled that of the normal control, with reduced bleeding; the therapeutic effects were comparable to those observed in the positive-control group. Effect of fusion proteins on ulcer index and inhibition rate Gastric mucosal injury of the protective model group was lower than that of the normal control group ( P < 0.0001). The inhibition rate of gastric ulcers of the two fusion protein intervention groups was 75.3% and 82.9% (Fig. 6 a), suggesting that the first dose of the protein relieves acute gastric mucosal injury caused by ethanol and also protects. In the treatment model, the inhibition rates of gastric ulcers for GGT-BPC157 and mScarlet-BPC157 were 84.6% and 80.4%, respectively ( P < 0.0001) (Fig. 6 b), suggesting that both proteins also benefit existing acute gastric ulcers. Histopathological changes of gastric mucosa in protective model rats HE staining and light microscopy were used to examine the gastric mucosa of rats (Fig. 7 ). In the Normal group, the mucosal layer was intact: the epithelial cells formed a continuous, non-shedding lining, glands were well organized, and no pathological changes were observed. By contrast, the Model group displayed extensive mucosal damage consistent with successful ulcer induction: over two-thirds of the mucosal epithelial cells exhibited degeneration and necrosis, and glandular architecture was markedly disrupted. Treatment with GGT-BPC157 markedly attenuated these lesions. Rats in this group showed substantially less epithelial shedding, an absence of edema and inflammatory cell infiltration, and largely preserved glandular structure with relatively regular arrangement, indicating that the fusion protein promotes protection and repair of the gastric mucosa. Recovery after mScarlet-BPC157 treatment was less pronounced than with GGT-BPC157 but remained clearly superior to the untreated Model group. In the mScarlet-BPC157 group, gland morphology was generally preserved, and inflammation was mild, suggesting that mScarlet-BPC157 also confers protective and reparative effects on gastric mucosa. Histological results showed that early administration of BPC 157 prevents gastric mucosal damage caused by ethanol. This protective effect against alcohol-induced gastric ulcers is due to its ability to reduce tissue damage, inhibit inflammation, and promote repair. Histopathological changes of gastric mucosa in the treatment model rats Figure 8 shows the histological sections of gastric mucosa of rats in each group: in the normal group, the mucosal layer is complete, the glands are evenly arranged, and the epithelial layer is continuous, without congestion, edema, and inflammatory cell infiltration, indicating that the gastric tissue function is normal. The model group showed typical pathological features of ethanol-induced gastric ulcer, including focal mucosal defect, congestion and edema, gland destruction, disorder of gland arrangement, and a large number of inflammatory cells infiltration. After treatment with the BPC 157 fusion protein, these pathological changes were obviously improved. Compared with the model group, the inflammation, congestion, and edema in the fusion protein group were alleviated, the ulcer area was reduced, the gland structure was more regular, and the mucosal structure was partially restored. These reparative effects were comparable to those observed in the positive standard group, which also demonstrated histological improvement. Taken together, the results indicate that the BPC 157 fusion protein mitigates alcohol-induced gastric mucosal injury and promotes ulcer healing with efficacy similar to the standard therapeutic agent. Histological results show that the fusion protein reduced structural damage and inhibited the inflammatory response. The effect of fusion protein on protecting against inflammatory factors in the gastric tissue of model rats To evaluate the inflammatory response in alcohol-induced gastric mucosal injury, we measured expression levels of the proinflammatory cytokines IL-6 (Fig. 9 a), TNF-α (Fig. 9 b), and IL-1β (Fig. 9 c) in gastric tissue from the protective model. The results are presented in the figure. As shown in the figure, concentrations of all three cytokines in the Model group were significantly higher than those in the Normal group. However, treatment with either fusion protein (GGT-BPC157 or mScarlet-BPC157) resulted in cytokine levels that were significantly lower than those in the positive control group. Both fusion proteins, GGT-BPC157 and mScarlet-BPC157, produced cytokine levels that were significantly lower than those of the positive standard product group. Notably, IL-1β, IL-6, and TNF-α concentrations showed highly significant decreases ( P < 0.0001). The decline in IL-1β is consistent with suppression of the early inflammatory response, whereas the decrease in IL-6 indicates attenuation of chronic inflammation. Downregulation of TNF-α suggests reduced direct injury to the gastric mucosa. Differences between the two fusion proteins were minimal, implying comparable anti-inflammatory efficacy. The effect of fusion protein on inflammatory factors in the gastric tissue of therapeutic model rats The treatment model shows the changes in each inflammatory factor. Normal rats had unchanged concentrations of IL-6 (Fig. 10 a), TNF-α (Fig. 10 b) and IL-1β (Fig. 10 c). However, the group receiving the fusion protein had significant reductions in all three inflammatory factors compared to the model group: IL-6 decreased 60% ( P < 0.0001), TNF-α decreased 65% ( P < 0.0001), and IL-1β decreased 55% ( P < 0.0001). The fusion protein inhibited more inflammatory factors than the positive standard group, which reduced IL-6 expression by about 50%, TNF-α expression by 55%, and IL-1β expression by 50%; thus, we conclude that the fusion protein reduces gastric mucosal inflammation by significantly downregulating IL-6, TNF-α, and IL-1β expression and is better than positive standards. Discussion It is difficult to produce bioactive peptides, on the one hand, because of its small molecular weight and easy degradation, and on the other hand, the expression system based on plasmid is unstable[ 32 ]. In order to solve these problems, the researchers transformed B. licheniformis 2709 into a production host. Unlike Escherichia coli, which is easy to form inclusion bodies, B. licheniformis is a strain of GRAS, and it also has strong secretory ability[ 33 ], so it is very suitable for producing secretory peptides. The researchers also modified the fermentation environment: knocking out the genes related to the synthesis of viscous extracellular polysaccharides, improving the rheological properties of the fermentation broth, and facilitating the subsequent separation and purification. In order to eliminate the separation instability and metabolic burden caused by the antibiotic screening plasmid, the researchers used CRISPR-Cas9 to integrate the expression cassette into the chromosome, so as to achieve genetically stable and antibiotic-free fermentation and meet the regulatory requirements of pharmaceutical and food-grade biological products[ 34 ]. The core of the expression strategy in this study is the design of fusion partners. Small peptides such as BPC 157, if expressed directly, are particularly vulnerable to rapid degradation by intracellular proteases. In order to protect the peptide from degradation, and at the same time, the researchers fused BPC 157 with GGT, and with the help of GGT's own efficient secretion signal, the apparent molecular weight of the peptide could be improved. In addition, the fusion with the fluorescent reporter protein mScarlet can also monitor the production and secretion process in real time and reliably. The analysis data also verified the rationality of these designs: the apparent molecular weight of GGT-BPC157 fusion protein increased slightly in SDS-PAGE electrophoresis, which is consistent with the common post-translation modifications (such as glycosylation) in secreted proteins of Bacillus[ 35 ]. Western blot analysis showed that the secreted fusion protein remained intact, indicating that the selected linker was stable in the extracellular environment. The experimental results show that carbon source and nitrogen source have a great influence on heterologous protein expression of Bacillus. The orthogonal experiment showed that glucose and soybean peptone were the key factors to increase the yield. Glucose has more advantages than compound starch, indicating that in this engineering strain, the easily available carbon source flux is efficiently used for protein production, rather than cell growth. In addition, soybean peptone may provide a balanced amino acid and small peptides as a precursor for the rapid synthesis of fusion protein. The fluorescence intensity is increased by three times, which also shows that it is feasible from molecular design to biotechnology, and statistical optimization is needed. The primary concern with fusion proteins is whether the appended partner alters the biological activity of the target [ 36 ]. In an ethanol-induced gastric ulcer model, in vivo data indicate that GGT-BPC157 and mScarlet-BPC157 exhibit therapeutic efficacy comparable to standard BPC 157 peptide, implying that N-terminal fusion does not disrupt BPC 157’s active site. Mechanistically, the recombinant peptides reduced proinflammatory cytokines (TNF-α, IL-1β, IL-6), indicating that the recombinant BPC 157 confers gastroprotection by modulating local inflammatory responses in a manner consistent with established cytoprotective pathways of native BPC 157. Histological analyses further demonstrate that the recombinant protein enhances gastric mucosal recovery, promoting tissue repair and restoring mucosal integrity [ 37 ]. In this paper, a simplified BPC 157 bio-manufacturing platform was built, which integrated strain modification, fermentation optimization, and a preliminary downstream purification process. Ammonium sulfate precipitation is a low-cost primary capture method, but industrial applications may require higher purity. Because the construct has a 6×His tag, nickel affinity chromatography (IMAC) can be used as a purification step to meet the purity requirements. In addition, the high expression level achieved in this study also provides the possibility for developing the strain into an oral veterinary drug biotherapy agent. In this application scenario, it is completely feasible to secrete BPC 157 in situ in the intestine. Declarations Consent for Publication Not applicable. Consent To Participate Not applicable. Funding This research was supported by the Beijing-Tianjin-Hebei Natural Science Foundation Cooperative Special Program (25JJJJC0023). Data Availability All data are given in the manuscript. Conflict of interest All authors declare that they have no conflict of interest. Author Contributions HZ and LS conceived and designed research. HZ, LS, QF, and QG conducted experiments.HZ contributed new reagents or analytical tools. QF analyzed data. HZ wrote the manuscript. All authors read and approved the manuscript. Ethical approval Approved by the Ethics Committee of Tianjin University of Science and Technology, all animal experiments conducted in this study strictly adhered to relevant ethical guidelines. Supplemental data Supplemental data including analysis of orthogonal experiments, construction of the integration vector, construction and screening of recombinant strain, effect of multiple factors on the fermentation of recombinant, optimization of fermentation conditions for recombinant, ammonium sulfate fractionation, precipitation of fusion proteins of these disruptants, are available online. References Akbarian, M., Khani, A., Eghbalpour, S., & Uversky, V. N. (2022). Bioactive peptides: Synthesis, sources, applications, and proposed mechanisms of action. International journal of molecular sciences , 23 , 1445. .https://doi.org/10.3390/ijms23031445 Peighambardoust, S. H., Karami, Z., Pateiro, M., & Lorenzo, J. M. (2021). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8706144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597218025,"identity":"df75fefb-40f9-43ce-ae57-725f4eb61151","order_by":0,"name":"Hanchao Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3QMUoEMRTG8RcCg0WGtBkQ9woPthBh0KvMsjDVFm43nYGRnCGDHmIrsczwJFXQVrBwwQtkO0sHW90Mdhb519+veA8gl/uPKQD67NRZAeRcxFpIqWcJIwj1UjK/Gm3XnlbWzRJO7LZdDX1Y0kmgGnWTFou7fk9bTWznAxIzLwLBsXjYHCfs3iMNj8Qx+GvamjdxzjWvhofjhKsNuDJQga+0o2EiF9oVvEyQ4psYEvi+RyrNs0DXpImYyLRsVaWfkERw80SpFkcbapTgm+nJa1HZsU/esrDrjxg7dWOAKEa8vJKyH+MhQX6L6b/tc7lcLvejL9keYcLSXrkRAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0006-7024-2699","institution":"Tianjin University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Hanchao","middleName":"","lastName":"Zhang","suffix":""},{"id":597218026,"identity":"61299680-aafa-43d5-a6cc-cc2dc6a0fd4c","order_by":1,"name":"Lanying Shao","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lanying","middleName":"","lastName":"Shao","suffix":""},{"id":597218027,"identity":"d72be1d8-a154-4c3a-9914-edfcedf26912","order_by":2,"name":"Qinghua Feng","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Feng","suffix":""},{"id":597218028,"identity":"2b46d658-4b91-4ce5-84c7-58f20c12b4a4","order_by":3,"name":"Qingping Guo","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qingping","middleName":"","lastName":"Guo","suffix":""},{"id":597218029,"identity":"ce1259e6-fb04-4ab6-a4a7-9d18f652e779","order_by":4,"name":"Haikuan Wang","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Haikuan","middleName":"","lastName":"Wang","suffix":""},{"id":597218030,"identity":"5acaf88f-e3e1-4dd9-a16f-4794bfe52a9f","order_by":5,"name":"Huitu Zhang","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huitu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-27 05:47:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8706144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8706144/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103857514,"identity":"a8f59016-9d4e-4435-8896-3519b1874270","added_by":"auto","created_at":"2026-03-03 18:46:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1618056,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the growth curve of the recombinant strain\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/efcfd57574286b4f4c42463d.png"},{"id":104400936,"identity":"62c4a023-4d4b-477a-bbd9-e3ebc6bb8871","added_by":"auto","created_at":"2026-03-11 12:11:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":738653,"visible":true,"origin":"","legend":"\u003cp\u003eFermentation time of recombinant strain \u003cem\u003eB.licheniformis\u003c/em\u003e 2709-mScarlet-BPC157\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/f1add30348b9c7a596e2e5c1.jpeg"},{"id":103857516,"identity":"06f3ce01-a0fa-4423-9f17-0ad779b72bb8","added_by":"auto","created_at":"2026-03-03 18:46:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1036267,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of the supernatant of the recombinant strain fermentation M: protein marker. \u003cstrong\u003ea\u003c/strong\u003eSDS-PAGE analysis of fermentation supernatants of recombinant strain GGT-BPC157 and control strain \u003cem\u003eB. licheniformis\u003c/em\u003e 2709. Lane 1: fermentation supernatants of GGT-BPC157; Lane 2: negative control (\u003cem\u003eB. licheniformis\u003c/em\u003e2709). \u003cstrong\u003eb\u003c/strong\u003e SDS-PAGE analysis of fermentation supernatants of recombinant strain mScarlet-BPC157 and control strain \u003cem\u003eB. licheniformis\u003c/em\u003e 2709. Lane 1: fermentation supernatants of mScarlet-BPC157; Lane 2: negative control (\u003cem\u003eB. licheniformis\u003c/em\u003e 2709).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/923fa91d11097f9c936e37f1.jpeg"},{"id":103857524,"identity":"19be9dcd-e67f-4c7c-a712-5dccd7da7a3e","added_by":"auto","created_at":"2026-03-03 18:46:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":900055,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot identification of recombinant fusion proteins GGT-BPC157 and mScarlet-BPC157\u003cstrong\u003ea\u003c/strong\u003e The Western blot gel results for the fermentation supernatants of the recombinant strains GGT-BPC157 and \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 show Lane 1 is the negative control (no bands), Lane 2 is the fermentation supernatant of the recombinant strain GGT-BPC157. \u003cstrong\u003eb\u003c/strong\u003e The Western Blot gel results for the fermentation supernatants of mScarlet-BPC157 and \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 show Lane 1 is the negative control (no bands), and Lane 2 is the fermentation supernatant of the strain mScarlet-BPC157.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/dfe721f721bcc8da68a217e6.jpeg"},{"id":104401568,"identity":"c5b7383d-c38c-4d08-99f3-e42be063d890","added_by":"auto","created_at":"2026-03-11 12:13:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":724744,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic appearance of gastric mucosa in the protection and treatment models \u003cstrong\u003ea\u003c/strong\u003eProtect the gastric anatomy of the model group; \u003cstrong\u003eb\u003c/strong\u003e Gastric anatomy in the treatment model group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/410db54112e6d6470061da5e.png"},{"id":103857515,"identity":"18252260-4109-457b-851c-b056fef2e426","added_by":"auto","created_at":"2026-03-03 18:46:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119801,"visible":true,"origin":"","legend":"\u003cp\u003eGastric ulcer inhibition rates of the protective model and the therapeutic model (\u003cem\u003en\u003c/em\u003e = 6). \u003cstrong\u003ea \u003c/strong\u003eInhibition rate of gastric ulcer in the protective model group; \u003cstrong\u003eb\u003c/strong\u003eInhibition rate of gastric ulcer in the treatment model group.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/aa2b298129b16e0a2dde623d.jpeg"},{"id":103857519,"identity":"f28a0be8-ade1-47bd-aa05-34ddc3f8f8f7","added_by":"auto","created_at":"2026-03-03 18:46:56","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2621669,"visible":true,"origin":"","legend":"\u003cp\u003eResults of HE staining in the stomach of protective model rats\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/d2a4cc10c16268b39bf1744a.jpeg"},{"id":103857518,"identity":"48879613-6695-40e1-9718-96fdfcb87c49","added_by":"auto","created_at":"2026-03-03 18:46:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1034669,"visible":true,"origin":"","legend":"\u003cp\u003eResults of HE staining of the stomach in each group of rats in the treatment model\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/6e0f34a108ee8c1a5a66fd5a.png"},{"id":104400820,"identity":"18c219f4-18ee-44ac-a209-ead655a46f1b","added_by":"auto","created_at":"2026-03-11 12:11:10","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1203793,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of IL-6, TNF-α, and IL-1β in each group of the protection model (\u003cem\u003en \u003c/em\u003e= 6) \u003cstrong\u003ea\u003c/strong\u003eExpression level of IL-6; \u003cstrong\u003eb\u003c/strong\u003e Expression level of TNF-α; \u003cstrong\u003ec\u003c/strong\u003eExpression level of IL-1β\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/dd2e1c6450670967b8b99068.jpeg"},{"id":103857523,"identity":"015ec011-8ef2-4b72-b92f-1eec01399c69","added_by":"auto","created_at":"2026-03-03 18:46:57","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1192713,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of IL-6, TNF-α, and IL-1β in each group of the treatment model (\u003cem\u003en \u003c/em\u003e= 6). \u003cstrong\u003ea\u003c/strong\u003eExpression level of IL-6; \u003cstrong\u003eb\u003c/strong\u003e Expression level of TNF-α; \u003cstrong\u003ec\u003c/strong\u003eExpression level of IL-1β\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/78591633f5e71e5776d61287.jpeg"},{"id":104779263,"identity":"a126db08-e804-4b3e-8337-39b983118bb5","added_by":"auto","created_at":"2026-03-17 07:37:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13154268,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/ff2c55bc-788d-4991-a691-a0b59cddf950.pdf"},{"id":104401429,"identity":"0b3eeec4-62b0-4c2b-8ba8-a0f0c9a92015","added_by":"auto","created_at":"2026-03-11 12:12:41","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":436046,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydataforthemanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8706144/v1/59fe57b7c2398368abfd043b.pdf"}],"financialInterests":"","formattedTitle":"Efficient Expression of Small Molecule Bioactive Peptides in Bacillus licheniformis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBioactive peptides can play a variety of physiological roles, such as antioxidation, antibacterial, and immunomodulation[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], so they have become a key research object in the field of drug research and development and functional food. At the same time, it can also be used as a growth additive[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and a feed additive, which can help improve nutrient absorption efficiency and maintain intestinal health[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCompared with macromolecular proteins, small peptides with a molecular weight of 1\u0026ndash;10 kDa have higher bioavailability and better stability, and can achieve therapeutic effects with lower dosage. Among these small peptides, BPC 157 (body protection compound\u0026thinsp;\u0026minus;\u0026thinsp;157) has attracted a lot of attention. It was first isolated from gastric juice. Although its molecular weight became smaller, it still retained the cytoprotective function of the parent protein[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is not afraid of hydrolysis in the stomach, but also can promote tissue repair of multiple organs from the gastrointestinal tract to soft tissue[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, BPC 157 can also regulate inflammatory reaction and promote angiogenesis, which is realized by interacting with the nitric oxide (NO) system[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical application prospect of BPC 157 is very good, but its industrial production still faces many bottlenecks. The chemical solid-state synthesis method has a high cost, but it also uses harmful solvents, and it is difficult to expand the production scale[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, heterologous biosynthesis is more sustainable and cheaper. However, the recombinant expression of short peptides often encounters the problems of intracellular protease degradation and low downstream purification efficiency[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Fusion of the target peptide with a stable carrier or functional label can improve the stability of the peptide and make the recovery process simpler.\u003c/p\u003e \u003cp\u003eChoosing the right host strain is the key to realizing efficient production. B. licheniformis is classified as a GRAS (generally considered safe) strain, which has two characteristics: it can efficiently secrete protein out of cells, and it can also grow by using cheap carbon sources[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. With the development of systems biology, B. licheniformis has become a multifunctional cell factory for producing enzymes and peptides[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe expression system based on a plasmid can achieve high yield, but it often brings a metabolic burden to the host, and there are also problems of separation instability. In addition, antibiotics cannot be used in food-grade products[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, integrating an expression cassette into a chromosome has obvious advantages, which can ensure genetic stability without screening pressure. If we combine the strong endogenous signal peptide and optimize the promoter strength through metabolic engineering[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the chromosome-encoded construct can achieve a reliable secretion level suitable for industrial fermentation.\u003c/p\u003e \u003cp\u003eThis paper introduces a simple biological process for producing BPC 157 in B. licheniformis. The researchers obtained stable strains through chromosome integration and fusion gene constructs, and also optimized fermentation parameters to improve the yield. In addition, the team developed an efficient purification process and evaluated the biological activity of the recombinant peptide through gastric mucosal protection experiments in vitro. These findings provide a method for large-scale biological production of BPC 157.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, plasmids, and reagents\u003c/h2\u003e \u003cp\u003eThe following strains were used in this study: \u003cem\u003eB. licheniformis\u003c/em\u003e 2709, maintained in our laboratory, served as the host for expressing fusion proteins. \u003cem\u003eEscherichia coli\u003c/em\u003e JM109 and \u003cem\u003eEscherichia coli\u003c/em\u003e EC135pM. Bam was obtained from the Institute of Microbiology, Chinese Academy of Sciences (IMCAS, Beijing, China) for plasmid construction and propagation. The shuttle vector pKSVT was supplied by Hubei University (Wuhan, China), while plasmid pTU-apr is part of our laboratory stock. PrimeSTAR Max DNA Polymerase and the restriction endonucleases SmaI, SacI, SpeI, and NotI were acquired from Takara Biomedical Technology (Beijing, China). Additionally, antibiotics such as kanamycin and spectinomycin hydrochloride were sourced from Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade and readily available.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA manipulation and strain construction\u003c/h3\u003e\n\u003cp\u003eThe coding sequence for BPC 157 was obtained from the Usp-BPC157 fusion entry in GenBank. Given that the choice of fusion partner significantly influences the secretion efficiency of recombinant peptides [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we selected GGT subunits (21\u0026ndash;25 kDa and 41\u0026ndash;65 kDa) to enhance the effective mass of the peptide [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We constructed a fusion protein that directly links BPC 157 to the GGT subunit, incorporating an N-terminal 6\u0026times;His tag. Design primers for amplifying the sequence encoding BPC 157 and subsequent validation(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the reporter assay, we fused the coding sequence of the red fluorescent protein mScarlet to BPC 157, also with an N-terminal 6\u0026times;His tag. All target sequences were synthesized by BGI (Beijing, China).\u003c/p\u003e \u003cp\u003eThe pKSVT-GGT-BPC157 vector was created by combining the GGT insert with the pKSVT shuttle vector. The mScarlet-BPC157 fusion fragment was amplified from the plasmid template using primers BR-F and BR-R. Homologous arms flanking the GGT gene were amplified from \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 genomic DNA with primer pairs GGT-UF/GGT-UR and GGT-DF/GGT-DR (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). The pKSVT vector underwent linearization through double digestion with SpeI and NotI (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), followed by the ligation of the homologous arms into the vector (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe products were transformed into \u003cem\u003eE. coli\u003c/em\u003e JM109. Verified plasmids were extracted and subsequently transformed into the methylation-proficient strain \u003cem\u003eE. coli\u003c/em\u003e EC135pM. Bam. Transformants were selected on plates containing kanamycin and spectinomycin, and plasmid methylation was initiated by the addition of \u003cem\u003eL\u003c/em\u003e-arabinose. The methylated plasmids were isolated and electroporated into competent \u003cem\u003eB.licheniformis\u003c/em\u003e 2709 cells, with transformants selected on kanamycin plates. Single-crossover integration was achieved by culturing at 42\u0026deg;C, followed by colony PCR (Fig. S2a). After incubation at 37\u0026deg;C in antibiotic-free medium, double-crossover events and plasmid backbone excision were conducted. The final integrated \u003cem\u003eGGT-\u003c/em\u003eBPC157 sequence was verified through DNA sequencing (Fig. S2b).\u003c/p\u003e \u003cp\u003eTo create the recombinant strain of mScarlet-BPC157 using the integration vector pTU-apr, the mScarlet-BPC157 fusion gene is cloned into pTU-apr, resulting in the recombinant plasmid pTU-apr-mScarlet-BPC157 (Fig. S3a). Subsequent steps involve methylation, electroporation, and allelic exchange, following the same procedures used for constructing the GGT-BPC157 strain (Fig. S3b). The final recombinant strain confirms the integration of \u003cem\u003emScarlet-BPC157\u003c/em\u003e at the genomic \u003cem\u003eaprE\u003c/em\u003e locus (Fig. S3c).\u003c/p\u003e\n\u003ch3\u003eDetermination of the growth curve of recombinant strains\u003c/h3\u003e\n\u003cp\u003eRecombinant strains were revived from glycerol stocks by streaking onto non-selective LB plates. After isolation of single colonies, they were cultured in 5 mL of LB broth at 37\u0026deg;C with shaking at 220 rpm for 12 h. Subsequently, 1 mL of the starter culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh LB medium. The optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was measured every 2 h over a 24 h period to monitor cell growth. The obtained absorbance values were utilized to generate a growth curve[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSDS-PAGE detection of fusion proteins\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eSDS-PAGE detection of fusion proteins\u003c/div\u003e \u003cp\u003eRecombinant strains were revived from glycerol stocks by streaking onto antibiotic-free LB agar and incubating inverted at 37\u0026deg;C overnight. Single colonies were inoculated into liquid LB and shaken at 220 rpm[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], 37\u0026deg;C for 12\u0026ndash;14 h. From this overnight culture, 1 mL was transferred into a 250 mL flask containing 50 mL LB and incubated until the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) reached 0.8\u0026ndash;1.0. This culture was then used as inoculum at (2% v/v) into the production medium and incubated under the appropriate conditions until protein expression reached its maximum. Cells were harvested by centrifugation at 10,000\u0026times; g for 20 min at 4\u0026deg;C, and the resulting supernatant was collected for downstream processing.\u003c/p\u003e \u003cp\u003eProtein samples were analyzed by SDS-PAGE following a three-step procedure. First, 20 \u0026micro;L of the supernatant was mixed with 5 \u0026micro;L of 5\u0026times; loading buffer and denatured at 100\u0026deg;C for 5 min [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Electrophoresis was carried out at 80 V through the stacking gel and then at 120 V through the resolving gel until the bromophenol blue dye front reached the bottom. Gels were washed with ultrapure water and stained using a rapid microwave-assisted Coomassie protocol: the gel was briefly boiled in water in the microwave for 15\u0026ndash;30 s, cooled in Coomassie Brilliant Blue solution with a short heating step, and then stained for 30 min. Finally, gels were destained in ultrapure water, replacing the water every 2 h until the background was clear.\u003c/p\u003e\n\u003ch3\u003eDetermination of mScarlet-BPC157 fusion protein expression levels\u003c/h3\u003e\n\u003cp\u003eShake-flask fermentations were carried out in 500 mL flasks using the sequence-verified recombinant strain \u003cem\u003eB. licheniformis\u003c/em\u003e (BL-apr-mScarlet-BPC157); a parental strain lacking the target genes served as the negative control. All fermentations were performed in biological triplicate. Samples were withdrawn every 12 h, and culture supernatants were collected and diluted as required. To quantify target protein expression, 200 \u0026micro;L of each supernatant was transferred to black, opaque 96-well plates (three technical replicates per biological sample). Fluorescence was recorded on a multifunctional microplate reader with excitation and emission wavelengths of 569 nm and 594 nm, respectively [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot verification of the fusion protein\u003c/h2\u003e \u003cp\u003eWestern blot was carried out on precast denaturing SDS-PAGE gels (Yisheng Biotechnology, Shanghai, China). Before use, the transfer buffer was chilled to -20\u0026deg;C. Proteins were then wet-transferred to a nitrocellulose (NC) membrane at a constant 200 mA for 50 min. Following the transfer, the membrane was washed on an orbital shaker with 1\u0026times; TBST for 5, 10, and 15 min sequentially, then blocked with 5% skim milk at room temperature (22\u0026ndash;25\u0026deg;C) for 60 min. After three additional TBST washes, the membrane was incubated with HRP-conjugated mouse anti-His tag antibody, washed again, and the protein bands were detected using ECL working solution and a chemiluminescence imaging system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimization of shake flask fermentation conditions for\u003c/b\u003e \u003cb\u003eBacillus licheniformis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe used the fluorescence intensity of the mScarlet fusion protein as a quantitative readout of recombinant protein production in BL-apr-mScarlet-BPC157 at the reported peak expression time [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Fluorescence measurements were performed after centrifugation and appropriate dilution to control for variations in medium composition and fermentation conditions. Initial screening experiments examined single-factor effects of carbon and nitrogen sources. On an equimolar basis, soluble starch, glucose, lactose, and sucrose each replaced the basal carbon source(cornmeal); optimal carbon source concentration was then evaluated across the range 48 to 96 g/L. For organic nitrogen sources, soybean peptone, soybean powder, soybean meal powder, yeast extract, and peanut meal powder were assessed at a constant total nitrogen content, with supplementation rates varied from 20 to 80 g/L[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Inoculum size was subsequently examined between 1% and 4% v/v using the identified optimal basal medium. Following single-factor screening, three influential variables\u0026mdash;carbon source concentration, nitrogen source concentration, and inoculum size\u0026mdash;were optimized in combination using an L\u003csub\u003e9\u003c/sub\u003e(3\u003csup\u003e3\u003c/sup\u003e) orthogonal array design. Each experimental condition was performed in triplicate. The original medium served as the negative control, and fluorescence intensity was used as the primary response variable.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAmmonium sulfate staged precipitation\u003c/h3\u003e\n\u003cp\u003eThe strain was cultivated in the optimized medium, and fermentation-broth samples were harvested at peak expression. Initial SDS-PAGE analysis was performed, after which the culture was centrifuged at 10,000\u0026times; g for 30 min, and the target protein was enriched by ammonium sulfate fractionation. The clarified supernatant was aliquoted into 20 mL portions, and saturated ammonium sulfate solution was added incrementally to achieve successive 10% saturation steps. Each increment was mixed by magnetic stirring for 5\u0026ndash;10 min and then held at 4\u0026deg;C for 1\u0026ndash;2 hours to allow precipitation. Precipitated material was collected by \u0026ldquo;salting out\u0026rdquo; followed by centrifugation at 10,000 RPM for 20 min; pellets were recovered and reconstituted in phosphate buffer. Both the supernatant and the reconstituted precipitate were transferred separately into dialysis bags and dialyzed at 4\u0026deg;C against 0.01 M PBS. To remove residual ammonium sulfate, the dialysis buffer was replaced 6\u0026ndash;8 times. To identify the optimal ammonium sulfate saturation range for primary recovery of the target protein, we analyzed both the dialyzed supernatant and pellet by SDS-PAGE. Remaining fermentation broth within the determined ammonium sulfate saturation range was processed to further concentrate the target protein [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Protein concentrations of the resulting fractions were measured by the BCA assay, and samples were stored at 4\u0026deg;C for subsequent studies on the prevention and treatment of gastric ulcers.\u003c/p\u003e\n\u003ch3\u003eEstablishment of the application model of fusion proteins in gastrointestinal inflammation\u003c/h3\u003e\n\u003cp\u003eA total of 90 healthy male Wistar rats (200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were selected and acclimated for one week at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃). After acclimation, rectal temperature was measured to establish baseline values.\u003c/p\u003e \u003cp\u003eMale Wistar rats (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;90) were acclimated for one week at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and baseline rectal temperatures were recorded. Two fusion proteins (GGT-BPC157 and mScarlet-BPC157) were prepared in 0.01 M PBS at 70 mg/mL, and standard BPC 157 was prepared at 10 ng/kg. Animals were assigned to protective and therapeutic experimental paradigms. For the protective model, 30 rats were randomly allocated into five groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 per group): Normal, Model, GGT-BPC157, mScarlet-BPC157, and Positive Control. Treatments were administered once daily by oral gavage (5 mL/kg) for 7 days; the Normal and Model groups received an equivalent volume of PBS. After the final dose, rats were fasted for 24 h. Gastric injury was then induced in all groups except the Normal control by intragastric administration of 95% ethanol (5 mL/kg). Animals were euthanized 4 h after gastric injury induction.In the therapeutic model, gastric injury was induced before treatment allocation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], after which rats were administered the treatment once daily for 7 days. Animals underwent a 24-hour fast before euthanasia.\u003c/p\u003e \u003cp\u003eMeasurements of gastric mucosal lesions were quantified by measuring the ulcer\u0026rsquo;s maximal longitudinal diameter (\u003cem\u003ed\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e) and maximal transverse width (\u003cem\u003ed\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e) with a vernier caliper; ulcer area was calculated as \u003cem\u003eS\u0026thinsp;=\u0026thinsp;π\u0026thinsp;\u0026times;\u0026thinsp;d\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e/2 \u003cem\u003e\u0026times; d\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e/2. Ulcer inhibition rate was then determined. Concentrations of TNF-α, IL-1β, and IL-6 in gastric tissue were measured by ELISA. Gastric specimens were processed, sectioned, and stained with hematoxylin-eosin (HE) for histological evaluation. Data were plotted and analyzed using GraphPad Prism 10 (GraphPad Software, Boston, MA, USA). Statistical comparisons employed one-way analysis of variance followed by t-tests. Significance thresholds were set as follows: 0.05\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 0.01\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Biochemical assays were performed by Tianjin Hongke Biotechnology Co., Ltd. (Tianjin, China).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the growth curve of recombinant strains\u003c/h2\u003e \u003cp\u003eGrowth curves illustrate bacterial proliferation under defined culture conditions. The recombinant strains \u003cem\u003eB. licheniformis\u003c/em\u003e (BL2709-GGT-BPC157) and \u003cem\u003eB. licheniformis\u003c/em\u003e (BL2709-mScarlet-BPC157) exhibit growth profiles that closely parallel the parent strain \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All three strains show low OD\u003csub\u003e600\u003c/sub\u003e values and slow growth during the initial culture period. In that early phase, the wild-type \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 increases in optical density slightly faster than the two recombinant strains. Approximately 16 h after inoculation, all strains enter the stationary phase. The OD\u003csub\u003e600\u003c/sub\u003e maxima of the two recombinants are comparable to one another but are marginally lower than the wild type. Thus, although the overall growth patterns remain essentially unchanged, the recombinant strains reach similar exponential-phase growth rates as the wild type while attaining a modestly lower final cell density in the stationary phase, likely reflecting the metabolic burden of heterologous gene expression. Because the fundamental growth dynamics are preserved, inocula for fermentation and optimization can be taken from the late logarithmic to early stationary phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFermentation of recombinant strains\u003c/h2\u003e \u003cp\u003eSingle colonies of the successfully constructed strains were inoculated into 5 mL LB and grown overnight at 37\u0026deg;C with shaking. Cultures were then added 1:50 into 50 mL of fresh LB (2% inoculum). When the culture reached OD\u003csub\u003e600\u003c/sub\u003e of 0.6\u0026ndash;0.8, cells were transferred into the fermentation medium at a 2% inoculum [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Previous work indicates that GGT activity peaks after 72 h of fermentation. Since the BPC 157 is fused to a subunit of GGT and has a relatively low molecular weight, the secretion level of the fusion protein was indirectly assessed by measuring GGT activity.\u003c/p\u003e \u003cp\u003eCorrectly sequenced strains and their parental controls were cultivated in the fermentation medium, and samples were collected every 12 h throughout the fermentation cycle to monitor extracellular fluorescence. Fluorescence increased progressively, reaching approximately 60,000 AU at 72 h, while the parental negative control showed no detectable signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicate that mScarlet-BPC157 was successfully expressed in \u003cem\u003eB. licheniformis\u003c/em\u003e and secreted into the medium. Because the gastric pentadecapeptide is fused to the red fluorescent protein, fluorescence intensity serves as a surrogate measure of the peptide\u0026rsquo;s expression and secretion. Using this proxy, the recombinant strain\u0026rsquo;s protein expression peaked at 72 hours of fermentation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE validation of recombinant strains\u003c/h2\u003e \u003cp\u003eShake-flask fermentations of the recombinant strains were carried out, and samples were collected at the secretion peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Following centrifugation (13,000\u0026times; g, 2 min), supernatants were recovered for analysis. Supernatants from the parental strain \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 grown under identical conditions served as the negative control. Protein expression was assessed by SDS-PAGE. The theoretical molecular weight of GGT-BPC157 is 23 kDa, yet the predominant band migrated near 25 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea); no corresponding bands appeared in the negative control, and the upward shift is consistent with post-translational modification. In contrast, mScarlet-BPC157 produced a single band at the expected molecular weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Collectively, these data indicate that both recombinant \u003cem\u003eB. licheniformis\u003c/em\u003e strains secreted their respective fusion proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot identification of recombinant fusion proteins\u003c/h2\u003e \u003cp\u003eThe expression of the recombinant fusion proteins was confirmed by Western blot targeting the C-terminal 6\u0026times;His tag. Immunodetection was performed using an HRP-conjugated mouse anti-His tag monoclonal antibody. The fermentation supernatant of the parental \u003cem\u003eB. licheniformis\u003c/em\u003e 2709 strain was used as a negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After electrophoretic separation and transfer to NC membranes, the antibody revealed distinct immunoreactive bands of GGT-BPC157 and mScarlet-BPC157. The absence of specific signals in the negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) confirmed the specificity of the detection. This shows that both His-tag fusion proteins were successfully expressed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe influence of the types and addition amounts of carbon sources on the fermentation of recombinant\u003c/b\u003e \u003cb\u003eBacillus licheniformis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCarbon source identity and concentration strongly influence recombinant bacterial fermentation because these substrates supply both the structural carbon and the energy required for growth and metabolism [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To quantify this effect, we examined how alternative carbon sources affected fermentation of \u003cem\u003eB. licheniformis\u003c/em\u003e, using the mScarlet fluorescence intensity as a proxy for recombinant protein expression. We selected corn flour (64 g/L) as the reference carbon source and compared it to soluble starch, glucose, lactose, and sucrose, each supplied at an equivalent carbon content. For each carbon source, experiments were run in triplicate. RFP fluorescence, measured from the culture supernatant, served as the readout for secretion efficiency and expression level. Carbon source type had a pronounced effect on protein expression (Fig. S4a). Glucose yielded the highest fluorescence, followed by sucrose and lactose; soluble starch and corn flour produced the lowest signals. The difference in fluorescence between glucose and corn flour was statistically significant, indicating that \u003cem\u003eB. licheniformis\u003c/em\u003e preferentially exploits mono- and disaccharides over polysaccharides for recombinant protein production. Based on these results, glucose was selected as the carbon source for subsequent experiments.\u003c/p\u003e \u003cp\u003eTo optimize the addition of glucose, we set four concentration gradients (48, 64, 80, and 96 g/L) were evaluated in triplicate. Results showed that fluorescence intensity increases slowly from 48 to 80 g/L. A peak was observed at 96 g/L, but the expression level at 80 g/L was slightly lower than that at 64 g/L.These results show that increasing glucose concentration can increase protein expression even if it is below the threshold. We chose 64, 80, and 96 g/L as carbon source levels for orthogonal experiments (Fig. S4b)\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe influence of the types and addition amounts of nitrogen sources on the fermentation of recombinant\u003c/b\u003e \u003cb\u003eBacillus licheniformis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNitrogen source type and concentration strongly affect fermentation performance and recombinant protein expression in bacteria because they determine the availability of nitrogenous precursors and thereby influence core metabolic activity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To quantify these effects, we evaluated the impact of several complex nitrogen sources on the fermentation of recombinant \u003cem\u003eB. licheniformis\u003c/em\u003e while keeping all other culture conditions constant. Glucose was fixed at 64 g/L as the carbon source. Using soybean meal powder as a control, four composite nitrogen sources (all with a concentration of 40 g/L) were tested: soybean peptone, soybean powder, yeast extract, and peanut meal powder. We measured expression of a red fluorescent protein reporter in the recombinant strains to compare how each nitrogen source modulated protein production. The type of nitrogen source has an obvious influence on the expression of the reporter gene (Figure S5a): the fluorescence intensity of the soybean peptone group is the highest, which indicates that it is most suitable to support the synthesis of recombinant protein under experimental conditions. The expression level of the soybean powder group was the second, and the fluorescence intensity of the yeast extract and peanut meal powder group was much lower. This result shows that even if the nitrogen content is the same, the source and formula of the compound nitrogen source are different, which will have different effects on the metabolic state of bacteria and the yield of recombinant protein.\u003c/p\u003e \u003cp\u003eBased on the results, soybean peptone was chosen as the best addition amount. Four concentration levels were set at 20, 40, 60, and 80 g/L. From 20 g/L to 40 g/L, fluorescence intensity increased gradually, peaked at 40 g/L, but decreased at 60 and 80 g/L. High nitrogen concentrations may hinder growth and protein expression (Fig. S5b). Soybean peptone in the appropriate amount can increase protein expression of recombinant bacteria. In orthogonal experiments, three supplementation levels (20 g/L, 40 g/L, 60 g/L) were chosen as nitrogen source levels.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe influence of inoculation size on the fermentation of recombinant\u003c/b\u003e \u003cb\u003eBacillus licheniformis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInoculation size directly affects the growth and metabolic efficiency of bacteria: insufficient inoculation size will prolong fermentation time and reduce yield[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while excessive inoculation size may inhibit production efficiency or metabolic activity. In order to quantify its influence on the synthesis of recombinant protein, the researchers compared four inoculation sizes of 1%, 2%, 3% and 4%(v/v) and detected the expression of fluorescent reporter protein. The fluorescence intensity increased with the increase of inoculation size, and the fluorescence of 1% inoculation group was significantly lower than that of other groups because of its low initial cell density and long delay period. The fluorescence intensity of the 4% inoculation group reached the peak (Figure S6). These results indicate that increasing the inoculation density can promote the growth of bacteria and increase the expression of target protein. Therefore, the orthogonal experiment selected 2%, 3% and 4% as the inoculation level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOrthogonal experiment\u003c/h2\u003e \u003cp\u003eSingle-factor optimization identified three fermentation variables\u0026mdash; inoculum size (A), soybean peptone concentration (B), and glucose concentration (C)\u0026mdash;each tested at three levels. We then organized an orthogonal experimental design using these factors and employed fluorescent protein expression intensity as the response variable. Range analysis of the resulting data ranked the factors by influence on protein expression as glucose (C) \u0026gt; soybean peptone (B) \u0026gt; inoculation volume (A) (Table S2). That analysis also indicated that Experiment 2 produced the highest aggregated expression level. Further Analysis of the mean values (\u003cem\u003eK\u003c/em\u003e), determined the optimal factor combination as A1B2C2, corresponding to an inoculum size of 2% (v/v), 40 g/L soybean peptone, and 80 g/L glucose (Fig. S7a). Fermentation carried out with this optimized medium yielded a fluorescence intensity roughly threefold greater than that obtained with the original medium, demonstrating that the optimized conditions substantially enhanced the recombinant strain\u0026rsquo;s protein expression capacity (Fig. S7b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAmmonium sulfate fractionation and precipitation of fusion proteins\u003c/h2\u003e \u003cp\u003eThe recombinant strain was cultivated in shake flasks using the optimized medium, and the culture broth was harvested at the time of maximal protein expression. Ammonium sulfate precipitation was carried out as described to precipitate GGT-BPC157 and mScarlet-BPC157. After precipitation, SDS-PAGE analysis was performed on both the supernatant and the precipitate. GGT-BPC157 began to precipitate at 40% ammonium sulfate saturation, but the resulting precipitate contained noticeable impurities. Raising the saturation to 50% caused most of the fusion protein to precipitate while reducing contaminating bands (Fig. S8a, b). In contrast, mScarlet-BPC157 did not begin to precipitate until 60% saturation; at this point, the target protein precipitated selectively, with relatively low levels of impurities. Increasing the saturation further to 90% precipitated most proteins present in the fermentation broth. Consequently, 50% and 60% saturation levels were identified as optimal for the purification of GGT-BPC157 and mScarlet-BPC157, respectively (Fig. S8c, d).\u003c/p\u003e \u003cp\u003eThe ammonium sulfate precipitation on large volumes of broth (optimal saturation) was resuspended with 0.02 M PBS and re-dissolved in a boiling EDTA (3.5 kDa) dialysis bag, 4\u0026deg;C, 0.02 M PBS, 24 hours. The dialysate was replaced until no flocculent precipitation was observed with 1% BaCl\u003csub\u003e2\u003c/sub\u003e. The fusion protein was measured using the BCA kit.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of fusion protein on the gastric morphology of ethanol-induced acute gastric ulcer in a rat model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConcentration of purified fusion protein measured with BCA kit (70 \u0026micro;g/mL) in 0.01 M PBS buffer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and administered according to the method. Gastric ulcer models were induced simultaneously in the protection and treatment groups. After modelling gastric mucosal morphology of rats in each group was assessed.\u003c/p\u003e \u003cp\u003eFigure results for the protection cohort are presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The gastric mucosa of the normal control group was intact and unremarkable, with no signs of congestion, edema, or structural damage. In contrast, the model group exhibited extensive injury, including cord-like or patchy hemorrhages, blackish-brown ulcers, and pronounced gastric bleeding. The BPC 157 positive-control group showed a clear therapeutic response. Both the GGT-BPC157 and mScarlet-BPC157 groups demonstrated only mild improvement, with sporadic ulcer foci remaining. Results for the treatment cohort are shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The normal group retained an intact, undamaged gastric inner wall, whereas the model group continued to display patchy hemorrhage and mucosal erosion. After treatment, gastric tissue in the experimental groups closely resembled that of the normal control, with reduced bleeding; the therapeutic effects were comparable to those observed in the positive-control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of fusion proteins on ulcer index and inhibition rate\u003c/h2\u003e \u003cp\u003eGastric mucosal injury of the protective model group was lower than that of the normal control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The inhibition rate of gastric ulcers of the two fusion protein intervention groups was 75.3% and 82.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggesting that the first dose of the protein relieves acute gastric mucosal injury caused by ethanol and also protects. In the treatment model, the inhibition rates of gastric ulcers for GGT-BPC157 and mScarlet-BPC157 were 84.6% and 80.4%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting that both proteins also benefit existing acute gastric ulcers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological changes of gastric mucosa in protective model rats\u003c/h2\u003e \u003cp\u003eHE staining and light microscopy were used to examine the gastric mucosa of rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the Normal group, the mucosal layer was intact: the epithelial cells formed a continuous, non-shedding lining, glands were well organized, and no pathological changes were observed. By contrast, the Model group displayed extensive mucosal damage consistent with successful ulcer induction: over two-thirds of the mucosal epithelial cells exhibited degeneration and necrosis, and glandular architecture was markedly disrupted. Treatment with GGT-BPC157 markedly attenuated these lesions. Rats in this group showed substantially less epithelial shedding, an absence of edema and inflammatory cell infiltration, and largely preserved glandular structure with relatively regular arrangement, indicating that the fusion protein promotes protection and repair of the gastric mucosa. Recovery after mScarlet-BPC157 treatment was less pronounced than with GGT-BPC157 but remained clearly superior to the untreated Model group. In the mScarlet-BPC157 group, gland morphology was generally preserved, and inflammation was mild, suggesting that mScarlet-BPC157 also confers protective and reparative effects on gastric mucosa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological results showed that early administration of BPC 157 prevents gastric mucosal damage caused by ethanol. This protective effect against alcohol-induced gastric ulcers is due to its ability to reduce tissue damage, inhibit inflammation, and promote repair.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological changes of gastric mucosa in the treatment model rats\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the histological sections of gastric mucosa of rats in each group: in the normal group, the mucosal layer is complete, the glands are evenly arranged, and the epithelial layer is continuous, without congestion, edema, and inflammatory cell infiltration, indicating that the gastric tissue function is normal. The model group showed typical pathological features of ethanol-induced gastric ulcer, including focal mucosal defect, congestion and edema, gland destruction, disorder of gland arrangement, and a large number of inflammatory cells infiltration. After treatment with the BPC 157 fusion protein, these pathological changes were obviously improved. Compared with the model group, the inflammation, congestion, and edema in the fusion protein group were alleviated, the ulcer area was reduced, the gland structure was more regular, and the mucosal structure was partially restored. These reparative effects were comparable to those observed in the positive standard group, which also demonstrated histological improvement. Taken together, the results indicate that the BPC 157 fusion protein mitigates alcohol-induced gastric mucosal injury and promotes ulcer healing with efficacy similar to the standard therapeutic agent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological results show that the fusion protein reduced structural damage and inhibited the inflammatory response.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of fusion protein on protecting against inflammatory factors in the gastric tissue of model rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the inflammatory response in alcohol-induced gastric mucosal injury, we measured expression levels of the proinflammatory cytokines IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), and IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec) in gastric tissue from the protective model. The results are presented in the figure. As shown in the figure, concentrations of all three cytokines in the Model group were significantly higher than those in the Normal group. However, treatment with either fusion protein (GGT-BPC157 or mScarlet-BPC157) resulted in cytokine levels that were significantly lower than those in the positive control group. Both fusion proteins, GGT-BPC157 and mScarlet-BPC157, produced cytokine levels that were significantly lower than those of the positive standard product group. Notably, IL-1β, IL-6, and TNF-α concentrations showed highly significant decreases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The decline in IL-1β is consistent with suppression of the early inflammatory response, whereas the decrease in IL-6 indicates attenuation of chronic inflammation. Downregulation of TNF-α suggests reduced direct injury to the gastric mucosa. Differences between the two fusion proteins were minimal, implying comparable anti-inflammatory efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of fusion protein on inflammatory factors in the gastric tissue of therapeutic model rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe treatment model shows the changes in each inflammatory factor. Normal rats had unchanged concentrations of IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea), TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb) and IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). However, the group receiving the fusion protein had significant reductions in all three inflammatory factors compared to the model group: IL-6 decreased 60% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), TNF-α decreased 65% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and IL-1β decreased 55% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fusion protein inhibited more inflammatory factors than the positive standard group, which reduced IL-6 expression by about 50%, TNF-α expression by 55%, and IL-1β expression by 50%; thus, we conclude that the fusion protein reduces gastric mucosal inflammation by significantly downregulating IL-6, TNF-α, and IL-1β expression and is better than positive standards.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is difficult to produce bioactive peptides, on the one hand, because of its small molecular weight and easy degradation, and on the other hand, the expression system based on plasmid is unstable[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In order to solve these problems, the researchers transformed B. \u003cem\u003elicheniformis\u003c/em\u003e 2709 into a production host. Unlike Escherichia coli, which is easy to form inclusion bodies, B. licheniformis is a strain of GRAS, and it also has strong secretory ability[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], so it is very suitable for producing secretory peptides. The researchers also modified the fermentation environment: knocking out the genes related to the synthesis of viscous extracellular polysaccharides, improving the rheological properties of the fermentation broth, and facilitating the subsequent separation and purification. In order to eliminate the separation instability and metabolic burden caused by the antibiotic screening plasmid, the researchers used CRISPR-Cas9 to integrate the expression cassette into the chromosome, so as to achieve genetically stable and antibiotic-free fermentation and meet the regulatory requirements of pharmaceutical and food-grade biological products[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The core of the expression strategy in this study is the design of fusion partners. Small peptides such as BPC 157, if expressed directly, are particularly vulnerable to rapid degradation by intracellular proteases. In order to protect the peptide from degradation, and at the same time, the researchers fused BPC 157 with GGT, and with the help of GGT's own efficient secretion signal, the apparent molecular weight of the peptide could be improved. In addition, the fusion with the fluorescent reporter protein mScarlet can also monitor the production and secretion process in real time and reliably. The analysis data also verified the rationality of these designs: the apparent molecular weight of GGT-BPC157 fusion protein increased slightly in SDS-PAGE electrophoresis, which is consistent with the common post-translation modifications (such as glycosylation) in secreted proteins of Bacillus[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Western blot analysis showed that the secreted fusion protein remained intact, indicating that the selected linker was stable in the extracellular environment.\u003c/p\u003e \u003cp\u003eThe experimental results show that carbon source and nitrogen source have a great influence on heterologous protein expression of Bacillus. The orthogonal experiment showed that glucose and soybean peptone were the key factors to increase the yield. Glucose has more advantages than compound starch, indicating that in this engineering strain, the easily available carbon source flux is efficiently used for protein production, rather than cell growth. In addition, soybean peptone may provide a balanced amino acid and small peptides as a precursor for the rapid synthesis of fusion protein. The fluorescence intensity is increased by three times, which also shows that it is feasible from molecular design to biotechnology, and statistical optimization is needed.\u003c/p\u003e \u003cp\u003eThe primary concern with fusion proteins is whether the appended partner alters the biological activity of the target [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In an ethanol-induced gastric ulcer model, in vivo data indicate that GGT-BPC157 and mScarlet-BPC157 exhibit therapeutic efficacy comparable to standard BPC 157 peptide, implying that N-terminal fusion does not disrupt BPC 157\u0026rsquo;s active site. Mechanistically, the recombinant peptides reduced proinflammatory cytokines (TNF-α, IL-1β, IL-6), indicating that the recombinant BPC 157 confers gastroprotection by modulating local inflammatory responses in a manner consistent with established cytoprotective pathways of native BPC 157. Histological analyses further demonstrate that the recombinant protein enhances gastric mucosal recovery, promoting tissue repair and restoring mucosal integrity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this paper, a simplified BPC 157 bio-manufacturing platform was built, which integrated strain modification, fermentation optimization, and a preliminary downstream purification process. Ammonium sulfate precipitation is a low-cost primary capture method, but industrial applications may require higher purity. Because the construct has a 6\u0026times;His tag, nickel affinity chromatography (IMAC) can be used as a purification step to meet the purity requirements. In addition, the high expression level achieved in this study also provides the possibility for developing the strain into an oral veterinary drug biotherapy agent. In this application scenario, it is completely feasible to secrete BPC 157 in situ in the intestine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent To Participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by the Beijing-Tianjin-Hebei Natural Science Foundation Cooperative Special Program (25JJJJC0023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e All data are given in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eHZ and LS conceived and designed research. HZ, LS, QF, and QG conducted experiments.HZ contributed new reagents or analytical tools. QF analyzed data. HZ wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Approved by the Ethics Committee of Tianjin University of Science and Technology, all animal experiments conducted in this study strictly adhered to relevant ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental data\u0026nbsp;\u003c/strong\u003eSupplemental data including analysis of orthogonal experiments, construction of the integration vector, construction and screening of recombinant strain, effect of multiple factors on the fermentation of recombinant, optimization of fermentation conditions for recombinant, ammonium sulfate fractionation, precipitation of fusion proteins of these disruptants, are available online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkbarian, M., Khani, A., Eghbalpour, S., \u0026amp; Uversky, V. N. (2022). 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Clopidogrel-induced gastric injury in rats is attenuated by stable gastric pentadecapeptide BPC 157. \u003cem\u003eDrug Design Development and Therapy\u003c/em\u003e, 5599\u0026ndash;5610. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DDDT.S284163\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S284163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus licheniformis, BPC 157, Chromosomal integration, Fusion protein, Fermentation optimization, Gastric ulcer","lastPublishedDoi":"10.21203/rs.3.rs-8706144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8706144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStable expression of γ-Glutamyltranspeptidase (GGT)-BPC157 and mScarlet-BPC157 in Bacillus licheniformis strain 2709 by chromosomal integration. Fermentation conditions were optimized using single-factor and orthogonal experiments to maximize yield. Under optimal conditions (2% inoculum, 40g/L soybean peptone, and 80g/L glucose), target protein titers increased threefold compared to the basal medium. The fusion proteins were purified by fractional ammonium sulfate precipitation; optimal saturations were 50% and 60%, respectively. Biological activity was assessed in a rat model of ethanol-induced acute gastric ulcer. Both proteins showed gastroprotective and therapeutic activity, with ulcer inhibition rates ranging from 75.3% to 82.9% protective regime and 80.4% to 84.6% therapeutic regime. Histological analysis showed that the treatment reduced mucosal inflammation and stimulated glandular repair. ELISA showed significant downregulation of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in gastric tissue. These results suggest a biotechnological production strategy for BPC 157 and applications for gastric ulcer treatment.\u003c/p\u003e","manuscriptTitle":"Efficient Expression of Small Molecule Bioactive Peptides in Bacillus licheniformis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 18:46:52","doi":"10.21203/rs.3.rs-8706144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-26T04:36:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T01:13:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2026-02-24T04:38:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T11:40:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2026-02-01T03:59:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e0ef3383-9ce5-49fc-8993-9c1113afd0b8","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T18:46:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 18:46:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8706144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8706144","identity":"rs-8706144","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}