Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis for Diabetes and Obesity Treatment

Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis for Diabetes and Obesity Treatment | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis for Diabetes and Obesity Treatment Ningyuan Ye , Francesco Di Pierro , Nicola Zerbinati , Maria Laura Tanda , Chenglong Duan , Jure Zupet , Jiahe Li doi: https://doi.org/10.1101/2025.10.07.680898 Ningyuan Ye 1 Department of Biomedical Engineering, College of Engineering and School of Medicine, University of Michigan , Michigan, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Francesco Di Pierro 2 Microbiota International Clinical Society , Turin, Italy 3 Scientific & Research Department, Velleja Research , Milan, Italy 4 Endocrine Unit, Department of Medicine and Surgery, University of Insubria , Varese, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicola Zerbinati 4 Endocrine Unit, Department of Medicine and Surgery, University of Insubria , Varese, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maria Laura Tanda 5 Department of Medicine and Technological Innovation, University of Insubria , Varese, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chenglong Duan 6 Qingdao Saiding Biological Pharmaceutical Co. , Shandong Province, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jure Zupet 7 Research and Development , Eustone, Slovenia Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jiaheli{at}umich.edu jure.zupet{at}eustone.eu Jiahe Li 1 Department of Biomedical Engineering, College of Engineering and School of Medicine, University of Michigan , Michigan, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jiaheli{at}umich.edu jure.zupet{at}eustone.eu Abstract Full Text Info/History Metrics Preview PDF Abstract Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and β-cell dysfunction. Glucagon-like peptide-1 (GLP-1) has emerged as a pivotal therapeutic target due to its roles in promoting insulin secretion, regulating appetite, and enhancing lipid metabolism. However, GLP-1-based therapies face significant challenges, including high production costs, complex delivery methods, and rapid in vivo degradation. To address these limitations, we developed a novel gut delivery strategy using Bacillus subtilis PY79 as a host for the expression of pentameric GLP-1. The genetically-modified strain, B. subtilis JH389, is demonstrated to maintain the same probiotic and antibiotic-sensitivity features belonged to the original strain PY79, and was deposited at the BCCM/LMG collection as B. subtilis LMG P-34037. The pentameric GLP-1 was designed to be digested by intestinal trypsin and to release active GLP-1 monomers capable of (i) resisting inactivativation by DPP-4 and trypsin, (ii) crossing the intestinal mucosa and (iii) entering the bloodstream. SDS-PAGE and Western blot confirmed the expression of pentameric GLP-1 in B. subtilis JH389, and fecal sample analysis demonstrated the strain gut engraftment. The administration of B. subtilis JH389 increased serum GLP-1 concentration, while also reducing blood glucose levels in a mouse model. Our in vitro and in vivo studies highlights the therapeutic potential of the JH389 strain and support the feasibility of using genetically engineered bacteria as a cost-effective and efficient delivery platform for GLP-1, paving the way for future applications in diabetes and metabolic disorder treatments. Introduction Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disease characterized by insulin resistance and impaired β-cell function, accounting for over 90% of global diabetes case. 1 In addition to genetic factors, such as TCF7L2 gene mutations, the majority of cases are closely associated with modern lifestyle factors, including high-calorie diets, physical inactivity, and chronic psychological stress. 2 , 3 These factors lead to insulin resistance and progressive β-cell dysfunction, ultimately resulting in hyperglycemia. 4 Glucagon-like peptide-1 (GLP-1) is an endogenous incretin secreted by intestinal L cells. 5 It plays a critical role in metabolic regulation, including promoting insulin secretion, delaying gastric emptying, suppressing appetite, and enhancing lipid metabolism, making it a pivotal target for diabetes treatment. 6 , 7 In recent years, GLP-1-based therapeutics, such as liraglutide and semaglutide, have demonstrated remarkable efficacy in controlling blood glucose and reducing body weight, establishing themselves as essential tools in diabetes management. 8 Additionally, GLP-1 has shown potential in improving non-alcoholic fatty liver disease, expanding its applications in metabolic disorders. 9 Various strategies have been explored to improve the stability and delivery efficiency of GLP-1 in therapeutic applications. One common approach is the conjugation of GLP-1 to macromolecules such as polyethylene glycol (PEG) or albumin, which increases its stability and extends its half-life by reducing enzymatic degradation. 10 – 14 Another method involves chemical encapsulation techniques, such as liposomes or polymer-based nanoparticles, which protect GLP-1 from degradation in the gastrointestinal tract and enable controlled release. 15 , 16 Additionally, microbial delivery systems using genetically engineered bacteria offer an innovative solution by allowing the in-situ production and secretion of GLP-1 in the gut. 17 – 20 Despite the widespread clinical use of GLP-1-based therapies, their high production costs and complex delivery methods limit their large-scale application. 21 – 23 Key challenges also include the susceptibility of GLP-1 to degradation by dipeptidyl peptidase-4 (DPP-4), resulting in a short half-life of only 1∼2 minutes. 24 , 25 Therefore, developing cost-effective, efficient delivery systems for GLP-1 formulations has become a critical area of research. Building on the pentameric strategy described in the study by Lin et al, where recombinant Lactobacillus was used for oral delivery of pentameric GLP-1 in diabetic rats, we adapted this approach for use in B. subtilis . 26 This adaptation leverages the robust genetic engineering potential and natural safety profile of B. subtilis , making it a versatile platform for microbial delivery systems. By employing molecular cloning techniques, we designed a pentameric GLP-1 expression system optimized for this host. The pentameric GLP-1 is engineered to resist enzymatic degradation during storage and production while being specifically cleaved by trypsin in the intestine to release active GLP-1 monomers. These monomers can subsequently cross the intestinal mucosa and enter the bloodstream to exert therapeutic effects. This study aims to refine and extend the pentameric delivery strategy to develop a low-cost, scalable, and efficient GLP-1 formulation for diabetes treatment and other metabolic disorders, addressing key limitations of current GLP-1-based therapies. Materials and methods 1. Materials All the materials are shown in Table 1 . All chemicals and reagents were purchased from Fisher Scientific International Inc. (New Hampshire, USA) unless otherwise noted, and were of highest purity or analytical grade. View this table: View inline View popup Table 1. Materials 2. Methods Homology-Directed Gene Editing The B. subtilis strain PY79 was selected for genetic editing. The amino acid sequence of the active form of GLP-1 was obtained from the NCBI database. After removing the last amino acid, the second amino acid A was mutated to G, and the 20 th and 28 th amino acids K were mutated to Q and D, respectively. This modified sequence was designated as trypsin-resistant GLP-1 (Tr GLP-1), while the unmodified sequence was referred to as wild-type GLP-1 (Wt GLP-1). Each sequence was repeated five times to construct pentameric GLP-1 sequences. The pentameric sequences were designated as Wt 5xGLP-1 for the unmodified sequence, and Tr 5xGLP-1 for the modified trypsin-resistant sequence. Additional variants were designed to include a Flag tag at the C-terminal end of the pentamer sequence. The sequences were then converted into corresponding nucleotide sequences, synthesized as gBlock double-stranded DNA fragments, and ordered from Twist Bioscience Corporation (South San Francisco, CA, USA). Similarly, the AmyE gene sequence was retrieved from NCBI, and primers were designed based on its upstream and downstream homologous regions. These primers were synthesized by IDT. PCR was used to amplify the homologous flanking regions and the GLP-1 gene sequence, and the reaction setup is listed in Table 2 . The three fragments were assembled using Gibson Assembly. 27 Competent cells of B. subtilis PY79 were then prepared using 1x MC medium, whose components are shown in Table 3 , and the assembled DNA fragments were transformed into the bacteria. The bacteria were plated on selective media containing the appropriate antibiotics. View this table: View inline View popup Download powerpoint Table 2. PCR reaction setup for fragment synthesis View this table: View inline View popup Download powerpoint Table 3. 1x MC medium setup of 100 mL Colony PCR Verification Single colonies of appropriate size and morphology were selected from the selective plates. After expanding the culture, genomic DNA was extracted and DNA fragments were amplified via PCR using sequencing primers verify the successful insertion of the GLP-1 sequence at the DNA level. Table 4 and Table 5 show the colony PCR reaction setup and running program. The amplified products were then sent for sequencing at Azenta Life Sciences (Massachussets, USA) with subsequent alignment analysis to additionally validate the genentic insertions. View this table: View inline View popup Download powerpoint Table 4. Colony PCR reaction setup View this table: View inline View popup Download powerpoint Table 5. PCR running program setup SDS-PAGE Electrophoresis Verification The SDS-PAGE preparation method is outlined in Table 6 . The bacterial culture supernatant, obtained after the transformation process, was subjected to precipitation and concentration using the TCA precipitation method. Proteins were denatured by heating after adding loading buffer. The prepared samples were loaded onto two identical polyacrylamide gels for electrophoresis. After electrophoresis, one gel was stained with Coomassie Brilliant Blue and destained overnight, while the other gel was used for membrane transfer. The membrane was subjected to immunoblotting using a Rat-derived anti-FLAG antibody and an HRP-conjugated Rabbit-derived anti-Rat antibody to confirm the successful expression of the pentameric GLP-1 at the protein level. View this table: View inline View popup Download powerpoint Table 6. SDS-PAGE gel setup Spore Preparation Using 2x SG Medium The 2x SG medium was prepared with components shown in Table 7 , with 200 µL of overnight bacterial culture spread onto each 2xSG agar plate. The plates were incubated at 30°C for 36 hours. The spore formation rate was examined under a microscope: if the rate exceeded 90%, the spores were scraped into sterile deionized water. The spores were then washed by repeated ultrasonication and centrifugation for three days. Finally, the spore suspension concentration was quantified via the serial dilution method. View this table: View inline View popup Table 7. 2x SG medium setup of 1 L Cre/lox-Mediated Marker Deletion Using pDR244 To remove the chloramphenicol (Cm) resistance cassette flanked by loxP sites at the amyE locus, we employed the Cre/lox recombination system. 28 The temperature-sensitive plasmid pDR244, encoding Cre recombinase and conferring spectinomycin (Spec) resistance, was introduced into the engineered Bacillus subtilis strain via transformation. Competent cells were prepared using 1× MC medium, and 500 ng of plasmid DNA was added to 400 μL of competent cells, followed by incubation at 30 °C. Transformants were plated on LB agar supplemented with Spec (50 μg/mL) to select for successful uptake of pDR244. A single Spec-resistant colony was inoculated into antibiotic-free LB broth and incubated at 42 °C overnight to induce Cre expression and simultaneously inhibit plasmid replication, facilitating its curing. The culture was then streaked onto LB agar to isolate single colonies, which were subsequently replica plated onto LB, LB with Spec, and LB with Cm plates. Colonies that grew only on antibiotic-free LB plates were considered candidate clones for marker excision and plasmid loss. Genomic DNA was extracted from these colonies, and PCR was performed using primers flanking the amyE locus and showing in the following sheet. The PCR product was sequenced to confirm complete deletion of the cat cassette and retention of a single loxP site. B. subtilis PY79 and B. subtilis JH389 isolation and purity check A small quantity of freeze-dried powder of each strain was streaked onto separate TSA (Tryptic Soy Agar; Oxoid SpA, Rodano, Milan, Italy) plates to isolate the two strains. The plates were incubated under aerobic conditions at 37°C for 48 hours. After incubation, a visual inspection confirmed the presence of a single colony morphology for both strains, as expected. One representative colony from each B. subtilis strain was subsequently picked, re-streaked on fresh TSA plates, and incubated to verify culture purity. Antibiotic resistance profiles Prior to performing the antibiotic susceptibility assay, B. subtilis PY79 and B. subtilis JH389 strains were propagated on TSA medium and incubated aerobically at 37°C for 48 hours. Minimal Inhibitory Concentrations (MICs) were determined for sixteen antibiotics: ampicillin, penicillin, clindamycin, linezolid (range: 0.03–16 μg/mL), vancomycin, ciprofloxacin (range: 0.25–128 μg/mL), neomycin, gentamicin, streptomycin (range: 0.5–256 μg/mL), kanamycin (range: 2–1024 μg/mL), erythromycin, quinupristin-dalfopristin (range: 0.016–8 μg/mL), tetracycline, chloramphenicol, rifampicin, and trimethoprim (range: 0.125–64 μg/mL), amoxicilline-clavulanate (range: 0.06–64 μg/mL), and cefixime (range: 0.5–64 μg/mL) using commercially prepared microplates (Sensititre™ EULACBI1 and EULACBI2, ThermoFisher). Following incubation, strain purity and viability were confirmed. Individual colonies were selected and resuspended in 3 mL of sterile saline solution. Cell suspensions were adjusted to a McFarland standard of 1.0, corresponding to approximately 3.0 × 10⁸ CFU/mL, then diluted 1:1000 in LSM broth. Within 30 minutes from preparation, 100 μL of the diluted bacterial suspension was dispensed into each well of the Sensititre™ microplates using a multichannel pipette. Negative control wells were inoculated with sterile LSM broth only. The Sensititre™ EULACBI1 and EULACBI2 plates contai ned predefined antibiotic concentrations according to ISO 10932:2010 guidelines. 29 Plates were incubated under aerobic conditions at 37°C for 48 ± 3 hours. Following incubation, negative control wells were examined for potential contamination. Provided that both positive and negative controls met quality criteria, MIC values were recorded. Resistance to simulated gastric juice The evaluation of resistance to simulated gastric juice (SGJ) was conducted following the internal method IM05, adapted from ISTISAN Reports 2008/36, with minor modifications. 30 B. subtilis PY79 and JH389 were first cultured in TSB (Tryptic Soy Broth; Oxoid SpA, Rodano, Milan, Italy) broth. For the assay, cultures were centrifuged, the supernatants discarded, and the resulting pellets resuspended in 5 mL of sterile SGJ (pH 3.4 ± 0.1). SGJ composition included sodium taurocholate (0.08 mM), phospholipids (0.02 mM), sodium (34 mM) and chlorine (59 mM) for a final pH of 3.4±0.1. The suspensions were incubated at 37°C, and 1 mL aliquots were withdrawn at three timepoints: T0, T30, and T60 minutes. Each sample was serially diluted and plated on TSA to determine total viable counts. Plates were incubated under aerobic conditions at 37°C for 48 hours. Results are expressed as two percentage values, corresponding to strain survival after 30 and 60 minutes of exposure to simulated gastric juice (SGJ), and are calculated using the formula P = (μ / M) * 100, where P represents the percentage of resistance of the microbial strain to SGJ; μ is the log₁₀-transformed viable cell count after 30 minutes (T30) or 60 minutes (T60) of incubation at 37°C in SGJ; and M is the log₁₀-transformed viable cell count at the initial timepoint (T0), immediately after resuspension in SGJ. This approach enables quantification of each strain’s tolerance to acidic stress over time. Resistance to simulated intestinal juice The evaluation of resistance to simulated intestinal juice (SIJ) were conducted using the in-house method IM06 which is accredited in accordance with ISO 17025:2017, with minor modifications. 30 , 31 The method is based on viable cell counts performed at defined timepoints: T0 (immediately after resuspension in the simulated intestinal juice, SIJ), T240 (after 240 minutes of incubation), and T360 (after 360 minutes of incubation) at 37°C. B. subtilis strains PY79 and JH389 were grown in TSB for 24 hours at 37°C under aerobic conditions. Cultures were centrifuged, the supernatants discarded, and the bacterial pellets resuspended in 5 mL of SIJ. At each timepoint (T0, T240, and T360), 1 mL of the suspension was collected, serially diluted, and plated on TSA for determination of viable counts. This allowed evaluation of the strains’ survival capacity under simulated intestinal conditions. SIJ composition included pancreatin (1 mM), ox-bile (3 mM) and sodium chloride (9 mM) for a final pH of 8±0.1. All plates were incubated for 48 hours at 37°C under aerobic conditions. Survival results were expressed as percentage values using the formula P = (μ / M) * 100 where P represents the percentage resistance of the microbial strain to simulated intestinal juice (SIJ); μ is the log₁₀-transformed viable cell count after 240 minutes (T240) or 360 minutes (T360) of incubation at 37°C in SIJ; and M is the log₁₀-transformed viable cell count at time zero (T0), immediately after resuspension in SIJ. This calculation provides a quantitative estimate of the strain’s resistance to intestinal conditions over time. Adhesion to HT29 cell lines The HT29 human intestinal epithelial cell line was routinely maintained in High Glucose DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 μg/mL L-glutamine, and 40 μg/mL gentamicin at 37°C in a 5% CO₂ atmosphere (all reagents from Euroclone; Pero, Milan, Italy). Two days prior to the adhesion assay, HT29 cells were rinsed with Hank’s Balanced Salt Solution (HBSS), detached using trypsin, counted, and diluted to 2.5×10⁵ cells/mL. One well per strain was seeded with the cell suspension in a 24-well plate, which was then incubated at 37°C with 5% CO₂ for 48 hours, until confluence was achieved. The day before the assay, B. subtilis PY79, B. subtilis JH389, and Lacticaseibacillus paracasei ATCC 334 (used as a technical control) were inoculated into TSB (for B. subtilis ) or MRS broth (for L. paracasei ) and incubated for 24 hours at 37°C under appropriate atmospheric conditions. On the day of the test, confluence of HT29 monolayers was confirmed. Wells were washed with HBSS and pre-incubated for 1 hour with 875 μL of DMEM High Glucose + 1% FBS at 37°C, 5% CO₂. For each sample, an additional well containing only 875 μL of medium (without cells) was included as a control. Meanwhile, the bacterial cultures were centrifuged and washed twice with sterile distilled water. Cell pellets were resuspended and adjusted to McFarland standard 0.5, corresponding to approximately 1.5×10⁸ CFU/mL. These suspensions were further diluted 1:10 in DMEM High Glucose + 1% FBS. A volume of 125 μL of the diluted bacterial suspension was added to each well (including controls), resulting in a Multiplicity of Infection (MOI) of 5:1 (bacteria:HT29 cells). The plates were incubated for 60 minutes at 37°C in a 5% CO₂ atmosphere. After incubation, the contents of the control wells (without cells) were collected, serially diluted, and plated onto appropriate media to determine the input CFU. In the wells with HT29 cells, the medium was removed, and monolayers were washed three times with 1 mL HBSS to eliminate non-adherent bacteria. To recover adhered bacteria, 100 μL of trypsin was added to each well and incubated for 5 minutes at 37°C. The resulting cell suspension was recovered using 900 μL of Maximum Recovery Diluent (MRD, Difco, BD), serially diluted, and plated for enumeration. Plates were incubated at 37°C under appropriate conditions, and viable counts were used to assess the adhesive capacity of each strain. Adhesion percentage (P) was calculated using the formula P=(μ /M)*100 where P is the adhesion percentage of the tested strain to HT29 cells; μ is the log-transformed viable count (CFU) of adhered bacteria; and M is the log-transformed viable count (CFU) of the corresponding strain in wells without HT29 cells. To validate the technical reliability of the assay, L. casei ATCC 334 was used as an internal control. This strain is considered acceptable if its adhesion percentage falls within the validated range of 64–71%, determined through repeated in-house testing. Mouse Oral Treatment A cohort of twenty mice was raised until they reached 8 weeks of age for oral administration. The mice were divided into two groups shown in Table 8 , with ten mice in each group. The first group served as the control group and was administered B. subtilis PY79 spores expressing the green fluorescent protein (Treatment GFP). The second group was administered PY79 spores expressing the trypsin-resistant pentameric GLP-1 (Treatment Tr 5xGLP-1). The administered strains contained a Cm resistance marker. The spore dosage per mouse was set at 1×10 8 CFU, and the suspension volume was 150 µL per mouse. Spores were directly delivered into the gastrointestinal tract using oral gavage needles. The spore gavage was repeated for three consecutive days, and mouse fecal samples were collected to verify colony formation and confirming successful colonization of the spores in the gastrointestinal tract. The mouse oral administration procedure was conducted in two trials on the same cohort of mice, with the second trial on the cohort commencing following mice recovery, and the removal of administered strain from the microflora. View this table: View inline View popup Download powerpoint Table 8. Mouse oral treatment experimental setup Blood Sampling and GLP-1 Serum Concentration Measurement The animal experiment was conducted in two trials. In the first trial, blood samples were collected from the orbital sinus on the third and fifth days following the start of oral administration. In the second trial, blood samples were collected on the third and seventh days. Blood cells were separated, and serum samples were sent for GLP-1 concentration analysis. Serum samples were were sent to Qingdao Saiding Biological Pharmaceutical Co., Ltd. for GLP-1 concentration analysis via liquid chromatography–tandem mass spectrometry. Glucose Tolerance Test After confirming successful colonization of the administered strains, glucose tolerance tests were performed on the third and fifth days following the start of oral administration in the first trial. All mice were fasted overnight by removing food at 6:00 PM the day prior. At 10:00 AM the following day, food was reintroduced, and blood glucose levels were measured two hours later with a glucose monitor kit. Results Homology-Directed Gene Editing Considering the natural high-efficiency DNA transformation capability of the B. subtilis PY79 strain, the replacement of part of the original AmyE gene sequence with GLP-1 can be achieved by synthesizing linear DNA fragments flanked with appropriate homologous sequences. We successfully amplified the upstream and downstream homologous sequences as well as the Wt and Tr GLP-1 sequences using primers. These three fragments were assembled into a single construct using Gibson Assembly and successfully transformed into the PY79 strain. Sequencing and alignment analysis confirmed the successful insertion of the target gene. Download figure Open in new tab Figure 1. Homology-Directed Gene Editing. (A) GLP-1 fragment is repeated five times and signal peptide is added to the front of the coding sequence for secretion. The versions of 5xGLP-1 with FLAG tag in the end are designed for easy detection by Western Blot. (B) Trypsin targets at the interval between two GLP-1 so that intact monomeric GLP-1 can be released after digestion. (C) Homologous arms for AmyE and the GLP-1 fragments were amplified, and Gibson assembly was used to connect the fragments into a new linear DNA construct, with expected amplified fragment sizes observed and used for transformation of the PY79 strain. Cre/lox-Mediated Deletion of Chloramphenicol Resistance Marker To enable future rounds of genome editing, we utilized the Cre/lox recombination system to remove the Cm resistance marker from the engineered B. subtilis strain. After transformation with pDR244, colonies resistant to Spec were successfully obtained, confirming plasmid uptake. Following overnight incubation at 42 °C in antibiotic-free LB, a portion of the resulting colonies lost both Spec and Cm resistance. When replica plating was performed, several colonies exhibited growth only on LB plates without antibiotics and failed to grow on either Spec or Cm plates. These results strongly suggested that the chloramphenicol gene had been excised by Cre recombinase and that pDR244 had been cured. To confirm this, PCR amplification of the amyE locus was performed using primers flanking the integration site, and the resulting products were sent for Sanger sequencing. Sequence analysis confirmed the successful deletion of the Cm resistance cassette, accurate retention of a single loxP site, and integrity of the adjacent genomic sequences. These findings confirm that the Cre/lox strategy using pDR244 enables clean and efficient removal of antibiotic resistance markers in B. subtilis , allowing for iterative genetic modifications without the accumulation of resistance cassettes. The resulting strain, designated JH389, contains no antibiotic resistance markers, while retaining genetic integrity of the expression cassette ( Figure 2 ). The JH389 strain was deposited at the BCCM/LMG collection as B. subtilis LMG P-34037. Download figure Open in new tab Figure 2. JH389 Expression Cassette. The expression cassette in the JH389 strains contains no Cm resistance marker following the Cre/lox-mediated excision. Verification of Pentameric GLP-1 Secretion After Cm Marker Excision by Protein Electrophoresis To confirm whether removal of the chloramphenicol resistance cassette affected secretion of the engineered pentameric GLP-1, we compared strains before and after Cm marker excision ( Figure 3 ). Both the original strain carrying the Cm marker and the Cre/lox-edited strain, JH389, were grown overnight in LB, and culture supernatants were collected for protein analysis. Proteins were precipitated using TCA, followed by SDS-PAGE. One gel was stained with Coomassie Brilliant Blue, while the other was subjected to Western blotting using an anti-FLAG antibody. Coomassie staining revealed distinct protein bands at the expected molecular weight (∼15 kDa) across all GLP-1-expressing lanes (highlighted in the red rectangle), indicating successful secretion of the target protein in both marker-retaining and marker-deleted strains. Western blot analysis detected clear signals for lanes containing FLAG-tagged constructs (lanes 1 and 3, marked by red arrows), confirming the specificity of FLAG detection. These findings demonstrate that the removal of the chloramphenicol resistance marker via Cre/lox recombination did not impair the ability of engineered B. subtilis strains to secrete pentameric GLP-1. This result also validates the compatibility of iterative genome editing using marker recycling with functional expression of heterologous proteins. Download figure Open in new tab Figure 3. Coomassie-Stained SDS-PAGE and Western Blot Analysis of Pentameric GLP-1 Secretion Before and After Cm Marker Excision. Left. Coomassie Brilliant Blue-stained SDS-PAGE showing TCA-precipitated supernatant proteins from different Bacillus subtilis strains. Red rectangle indicates the expected position (∼15 kDa) of pentameric GLP-1. Right. Western blot detection using anti-FLAG antibody. Red arrows indicate FLAG-tagged GLP-1 constructs. Lane assignment (left to right): 1. Wt 5xGLP-1 Flag 2. Wt 5xGLP-1 3. Tr 5xGLP-1 Flag 4. Tr 5xGLP-1 5. Tr 5xGLP-1 (Cultivated with LB containing Cm) 6∼8. Tr 5xGLP-1 (Cm marker excised). B. subtilis strains isolation and purity check A colony of each B. subtilis strains was recovered and streaked to assess the purity check verifications ( Figure 4 ). A single B. subtilis-typical morphology was recovered in the plates, indicating strain purity. Download figure Open in new tab Figure 4. Strain Isolation and Purity Check. Strains a) B. subtilis PY79, and b) B. subtilis JH389 (in the figure indicated as LMG P-34037), were streaked onto TSA agar plate. Only one morphology was recovered in the plates displaying the typical shape of the B. subtilis strain. Antibiotic resistance profiles Antibiotic resistance profiles of both B. subtilis strains are listed in Table 9 . Globally, the antibiotics tested exhibited minimum inhibitory concentrations (MICs) equal to or below the microbiological cut-off values for Bacillus subtilis , as defined by the European Food Safety Authority (EFSA). The only exception was observed for streptomycin, where the B. subtilis JH389 strain showed a two-fold increase and the PY79 strain exhibited a four-fold increase over the cut-off. It is worth noting that two-fold deviations are commonly observed in MIC testing and are generally considered acceptable by EFSA due to natural biological variability. Based on these results, B. subtilis JH389 can be regarded as safe with respect to antibiotic resistance. View this table: View inline View popup Table 9. MIC values (expressed as µg/mL) for B. subtilis JH389 and B. subtilis PY79. Resistance to simulated gastric juice In Table 10 are reported the survival data, expressed as Log (CFUs), obtained from the simulated gastric transit resistance assay for the two B. subtilis strains under investigation. The percentage of residual viability at 30 and 60 minutes of exposure to simulated gastric juice confirmed that both B. subtilis strains exhibited high tolerance to acidic conditions. Specifically, B. subtilis PY79 retained 99.4% and 98.4% viability at T30 and T60, respectively, while B. subtilis JH389 showed 99.1% and 97.8% residual viability at the same timepoints, indicating comparable gastric resistance. View this table: View inline View popup Table 10. Viable cell counts Log(CFUs) of the two tested strains following exposure to simulated gastric juice (SGJ). Resistance to simulated intestinal juice (SIJ) In Table 11 are reported the survival data, expressed as Log(CFUs), obtained from the simulated intestinal transit resistance assay for the two B. subtilis strains under investigation. The percentage of residual viability at 240 and 360 minutes of exposure to simulated intestinal juice confirmed that both B. subtilis strains exhibited high tolerance to pancreatin and bile. Specifically, B. subtilis PY79 retained 81.2% and 79.1% viability at T240 and T360, respectively, while B. subtilis JH389 showed 84.4% and 81.9% residual viability at the same timepoints, indicating comparable intestinal resistance. View this table: View inline View popup Table 11. Viable cell counts Log(CFUs) of the two tested strains following exposure to simulated gastric juice (SGJ). Adhesion to HT29 cell lines In Table 12 are reported the adhesion percentages, obtained on HT29 cell line for the two B. subtilis strains under investigation. Their adhesion capacity was evaluated using the HT29 human intestinal epithelial cell line. The technical control strain, L. paracasei ATCC 334, displayed an adhesion percentage of 68%, which falls within the experimentally validated range of 64–71%, thereby confirming the robustness of the assay. Both B. subtilis PY79 and JH389 showed measurable adhesive capacity to HT29 cells, with adhesion percentages of 61% and 66%, respectively. Fluctuations in adhesive properties within a ± 5% range are considered physiologically normal and fall within the expected variability of the assay. Therefore, adhesion percentages within this interval should be regarded as comparable. View this table: View inline View popup Download powerpoint Table 12. Adhesion assay results on HT29 cell line. Mouse Oral Treatment At 8 weeks of age, the two groups of mice underwent oral gavage. Fecal samples from both groups of mice were collected, resuspended, and plated on Cm-selective plates. The results in Figure 5 confirmed that the administered spores transitioned into vegetative cells and successfully colonized the mouse intestinal tract. Download figure Open in new tab Figure 5. Mouse Oral Treatment. (A) Mice were treated 8 weeks after birth. Oral gavage was performed for three days consecutively with either PY79 GFP spores, or PY79 Tr 5xGLP-1 spores. Blood collection started following the conclusion of the three day gavage. The blood was collected on day 3 and day 5 in the first trial, and at day 3 and day 7 in the second trial. Glucose tolerance tests were conduct at day 3 and day 5 in the first trial. (B) Fecal samples collected from GFP (top plate) and Tr 5xGLP-1 (bottom plate) treatment groups were spread onto the selective LB agar plates with chloramphenicol. Blood Sampling and Measurement of Serum GLP-1 Concentration Blood samples were collected, mixed with anticoagulants, and centrifuged to separate serum from blood cells. The serum samples were then analyzed for the concentration of GLP-1 ( Figure 6 ). Download figure Open in new tab Figure 6. Measurement of Serum GLP-1 Concentration. Blood samples from the mice were collected to quantify the concentration of monomeric GLP-1 in serum. (A) In the first trial, blood samples from all the groups were collected at days 3 days and 5 days after the beginning of gavage. The group administered Tr 5xGLP-1 showed an average concentrations of GLP-1 at 20.61 nM on day 3 and 23.96 nM on day 5. (B) In the second trial, blood samples from all the groups were collected at 3 days and 7 days after the beginning of gavage. The group administered Tr 5xGLP-1 showed an average concentrations of 19.75 nM on day 3 and 29.27 nM on day 7. In both trials, mice administered GFP spores showed almost no detectable GLP-1 in serum. In the first trial, the second group treated with Tr 5xGLP-1, showed an average GLP-1 concentration of 20.61 nM on day 3 and 23.96 nM on day 5. In the second trial, the second group showed an average GLP-1 concentration of 18.72 nM on day 3 and 29.27 nM on day 7. In contrast, the control group, treated with GFP spores exhibited almost no detectable GLP-1 in serum. The significant difference in GLP-1 concentration indicates that the GLP-1 detected in the serum of mice gavaged with Tr 5xGLP-1 spores is not endogenous but is almost entirely secreted by the colonized B. subtilis expressing the trypsin-resistant pentameric GLP-1. Thus, the secreted GLP-1 in the intestine is digested by the pancreatic trypsin into multiple active GLP-1 monomers, which subsequently cross the intestinal mucosal barrier and enter the bloodstream. Glucose Tolerance Test The mice treated with Tr 5xGLP-1 expressing spores exhibited lower blood glucose levels two hours after feeding, compared to the control group treated with GFP expressing spores ( Figure 7 ). This indicates that trypsin-resistant pentameric GLP-1 expressed by the transformed bacterial strains effectively exerts a hypoglycemic effect after crossing the intestinal mucosa and entering the bloodstream. Download figure Open in new tab Figure 7. Glucose Tolerance Test. Mice were fed after fasting for 16 hours. Two hours after feeding, blood samples were collected from tails and a glucose monitoring kit was used to determine the level of blood glucose. Discussion In this study, starting from B. subtilis PY79, we successfully constructed a strain, B. subtilis JH389 capable of expressing pentameric GLP-1, with increased resistance to trypsin and DPP-4 proteolysis, and demonstrated that the secreted pentameric GLP-1 can be digested by trypsin in the intestine into multiple active GLP-1 monomers, which then cross the intestinal mucosa and enter the bloodstream to exert their biological effects. Compared to the traditional method of exogenous GLP-1 injection, this study proposes a cost-effective gut delivery strategy based on genetically engineered bacteria, which not only reduces the inconvenience of repeated injections for patients but also provides a novel approach for the treatment of diabetes and obesity. The chromosomial modifications inserted into the AmyE gene of B. subtilis PY79, that gave rise to the strain B. subtilis JH389, did not change the probiotic features of original strain, including antibiotic resistance profiles, survival under simulated gastrointestinal conditions and adhesive capacity to human intestinal epithelial cells. B. subtilis JH389 can be then considered safe, as the observed two-fold increase in MIC for streptomycin falls within the variability generally tolerated by EFSA. In contrast, in our experimental conditions, the PY79 strain exhibited a four-fold increase for the same antibiotic. As regards to cefixime, some authors have described Bacillus strains with MICs 32 µg/mL as resistant. Both strains, therefore, demonstrate an intermediate phenotype but shifted toward sensitivity. Several factors could be responsible for this phenotype, for example, genes encoding beta-lactamases, which are inducible, or mechanisms linked to proteins capable of binding the antibiotic, or increased resistance to stress and genes acquired through ESBLs. 32 Both strains demonstrated high resistance to simulated gastric juice, maintaining over 97% residual viability after 60 minutes of exposure. Similarly, during exposure to simulated intestinal juice, the strains retained substantial viability over extended incubation periods (240 and 360 minutes), confirming their robust tolerance to gastrointestinal transit. In adhesion assays using the HT29 human intestinal epithelial cell line, B. subtilis PY79 and the strain JH389 exhibited comparable adhesion percentages. These results indicate that the genetically-modified B. subtilis JH389 (LMG P-34037) possesses essential probiotic traits supporting its potential application as probiotic candidate. Our previous experiments showed that the genetically-modified PY79 strain directly expressing GLP-1 monomers or exenatide failed to yield detectable expression in in vitro assays. This might be attributed to the low stability and high susceptibility to degradation of small peptides in prokaryotic expression systems, highlighting a limitation of prokaryotic platforms for small peptide expression. However, the strategy of expressing the product in a multimeric form and subsequently digesting it in the target environment effectively overcomes this issue. This delivery mode is not only applicable to GLP-1 but also provides valuable insights for future studies. For example, functional peptides such as antimicrobial peptides, signaling peptides, and anticancer peptides could also be designed in multimeric forms to enhance their expression efficiency in prokaryotic systems while ensuring gradual release of active monomers in vivo . We plan to further optimize this system and evaluate its effectiveness in disease models. Specifically, we will focus on an obese mouse model to comprehensively validate the strain’s effects on alleviating obesity and reducing blood glucose levels. Experimentally, we will perform continuous gavage administration over several days, followed by weekly glucose tolerance tests. Blood glucose levels will be measured at multiple time points after glucose administration to assess glucose responsiveness. Additionally, changes in food intake, body weight, and body fat percentage will be monitored to evaluate the strain’s impact on obesity. Furthermore, organ weight, insulin levels, and liver and kidney function will be analyzed to investigate the broader metabolic effects of exogenous GLP-1 in improving glucose and lipid metabolism and protecting organ function. Our future research will focus on five main aspects. First, enhancing precision delivery. Indeed, by leveraging the characteristics of B. subtilis , specific regulatory elements can be designed through genetic editing to enable controlled release of GLP-1. For instance, temperature-sensitive or pH-sensitive release systems could be developed to maximize strain efficacy in specific intestinal environments. Second, exploring combination therapies. By integrating multiple functional proteins (e.g., GLP-1, anti-inflammatory factors, or appetite suppressants) into a single strain, multi-target combination therapies could be developed to investigate their synergistic effects on metabolic diseases. Third, expanding spore delivery platform functionality. B. subtilis spores could be further utilized as a vaccine delivery platform by expressing specific antigens on the spore surface. Through controlled conversion mechanisms, antigen release timing could be optimized to avoid excessive immune responses. Additionally, small molecule drugs or nucleic acid therapeutics could be loaded onto the spore surface, enabling cross-disciplinary therapeutic applications. Fourth, engineering strain stability. Future studies should focus on the long-term stability and controllability of the strain in vivo . For example, biosafety designs could be implemented to ensure the engineered strain self-eliminates after fulfilling its therapeutic role, thereby minimizing potential environmental risks. Fifth, considering clinical translation. To achieve clinical applicability, it is essential to develop scalable and human-compatible production processes and quality control protocols. Using food-grade B. subtilis as a base carrier and veryfing its safety profile by subjecting the genetically-modified strain to mutagenesis, acute and sub-chronic toxicological investigations, activity currently ongoing according to the OECD (Organization for Economic Co-operation and Development) guidelines and in GLP-recognized laboratories, could ensure safety and controllability in human applications. In conclusion, the delivery strategy developed in this study holds significant promise for diabetes treatment, while also providing important insights for the biological therapy of various diseases, including metabolic, cardiovascular and neurological. 33 , 34 In future research, we aim to introduce more sophisticated regulatory mechanisms and multifunctional designs to enhance the practicality and translatability of this system, thus opening new avenues for synthetic biology in medical applications. 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Share Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis for Diabetes and Obesity Treatment Ningyuan Ye , Francesco Di Pierro , Nicola Zerbinati , Maria Laura Tanda , Chenglong Duan , Jure Zupet , Jiahe Li bioRxiv 2025.10.07.680898; doi: https://doi.org/10.1101/2025.10.07.680898 Share This Article: Copy Citation Tools Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis for Diabetes and Obesity Treatment Ningyuan Ye , Francesco Di Pierro , Nicola Zerbinati , Maria Laura Tanda , Chenglong Duan , Jure Zupet , Jiahe Li bioRxiv 2025.10.07.680898; doi: https://doi.org/10.1101/2025.10.07.680898 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Bioengineering Subject Areas All Articles Animal Behavior and Cognition (7640) Biochemistry (17706) Bioengineering (13902) Bioinformatics (41978) Biophysics (21465) Cancer Biology (18611) Cell Biology (25528) Clinical Trials (138) Developmental Biology (13387) Ecology (19920) Epidemiology (2067) Evolutionary Biology (24332) Genetics (15615) Genomics (22519) Immunology (17747) Microbiology (40424) Molecular Biology (17194) Neuroscience (88662) Paleontology (667) Pathology (2838) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)