The rising prevalence of obesity necessitates novel therapeutic strategies. Probiotics, particularly Lactobacillus species, have shown potential in weight management, yet the specific molecular mechanisms remain largely unknown.
This project aimed to identify and validate a specific anti-adipogenic component from Lactobacillus using a systematic Design-Build-Test-Learn (DBTL) engineering approach.
1. We screened six Lactobacillus strains and discovered that only the supernatant and exosomes from Lactobacillus rhamnosus significantly inhibited lipid accumulation in 3T3-L1 adipocytes, with exosomes showing a remarkable 80% reduction.
2. Proteomic analysis of these potent exosomes identified the Imidazole glycerol phosphate synthase subunit (hisF) as a highly abundant protein candidate.
3. Subsequently, the hisF gene was cloned, and the protein was expressed and purified.
4. Functional assays confirmed that the purified hisF protein alone inhibited adipogenesis by up to 50% in a dose-dependent manner.
5. Mechanistic studies revealed that hisF exerts its effect by up-regulating the master metabolic regulator AMPK, which in turn down-regulates the key adipogenic transcription factors PPARγ and C/EBPα.
Our findings provide a clear proof of concept, identifying the exosomal protein hisF from L. rhamnosus as a novel, potent inhibitor of adipogenesis and a promising candidate for future anti-obesity therapeutics.
Figure 1. Abstract figure of project description
The Escalating Global Obesity Crisis
The world is facing an unprecedented obesity pandemic.[1] According to the World Health Organization, over one billion people globally are living with obesity, a number that continues to climb at an alarming rate.[2] This condition is far more than a matter of weight; it is a complex, chronic disease that serves as a major risk factor for a host of debilitating secondary conditions.[3] These include type 2 diabetes, cardiovascular diseases such as heart attack and stroke, various forms of cancer, and musculoskeletal disorders.[4] The immense strain that obesity and its related illnesses place on global healthcare systems, coupled with the profound impact on individuals’ quality of life, underscores the urgent need for more effective and accessible therapeutic interventions.[5]
Figure 2. The escalating issue of obesity
The Shortcomings of Current Pharmaceutical Solutions
GLP-1 receptor agonists, such as semaglutide (Wegovy), have shown marked efficacy in weight reduction but remain limited by important drawbacks.
Adverse Effects: Common gastrointestinal symptoms (nausea, vomiting, diarrhea, constipation) frequently impair adherence. More serious risks, including gallbladder disease, pancreatitis, and disproportionate loss of lean body mass, have also been reported.[6][7]
Economic Barriers: Monthly costs often exceed $1,300, restricting accessibility and scalability.[8][9]
Weight Regain: Substantial weight regain after treatment discontinuation underscores their role as long-term management rather than curative therapy.[10][11]
Safety Concerns: Long-term safety data remain insufficient, with potential cumulative risks yet to be fully clarified.[12]
Figure 3. Current problems of solution
Our Solution: Engineering a Probiotic-Derived Protein for Targeted Therapy
We developed a novel anti-obesity strategy by harnessing a probiotic-derived protein through synthetic biology. Screening six Lactobacillus strains revealed Lactobacillus rhamnosus as the most effective in reducing lipid accumulation. Exosome analysis demonstrated ~80% inhibition of fat storage, and proteomics identified the key protein, hisF.
We cloned, expressed, and purified recombinant hisF, which alone suppressed lipid accumulation by up to 50% in a dose-dependent manner. Mechanistically, hisF activates AMPK, thereby downregulating PPARγ and C/EBPα, halting adipogenesis.[13]
This engineered hisF protein represents a targeted, safer, and sustainable therapeutic candidate that aligns with natural metabolic regulation to combat obesity.
| Feature | Our Microbiome Therapy (hisF Protein) | Wegovy (Semaglutide) |
|---|---|---|
| Mechanism of Action | Activates the body’s natural “metabolic master switch” (AMPK) to block fat cell formation. | Mimics a gut hormone (GLP-1) to suppress appetite centrally in the brain. |
| Source | Naturally derived protein from a beneficial probiotic, Lactobacillus rhamnosus. | Synthetic peptide analog, manufactured chemically. |
| Specificity | Highly targeted to the molecular pathway of adipogenesis. | Acts systemically on GLP-1 receptors throughout the body (brain, gut, pancreas). |
| Potential Side Effects | Anticipated to be minimal, leveraging a natural biological pathway. | High incidence of severe gastrointestinal issues (nausea, vomiting, etc.). |
| Cost & Accessibility | Potentially low-cost production via standard protein expression systems. | Extremely expensive (>$1,300/month), making it inaccessible for many. |
| Sustainability of Effect | Aims to modulate a core metabolic process for potentially more lasting effects. | Requires continuous weekly injections; weight is often regained after stopping. |
Table 1. Comparison table of microbiome therapy and Wegovy
Figure 4. Schematic figures of our solution: microbiome therapy
Wet Lab Experiments
Our project was brought to life through a structured Design-Build-Test-Learn (DBTL) cycle, using the 3T3-L1 pre-adipocyte cell line as our primary model for fat cell differentiation called adipogenesis.
Screening for Anti-Adipogenic Activity: We began by culturing six different species of Lactobacillus. We performed co-culture experiments with these live bacteria and differentiating 3T3-L1 cell and treat gentamycin 24hours to kill excessive growth of bacteria. Using Oil Red O staining to quantify lipid accumulation and identify the most potent anti-adipogenic strain.[14]
Isolation of the Active Component: To determine if the effect was caused by a secreted factor, we collected and tested the culture supernatant from each strain. After identifying the supernatant from Lactobacillus rhamnosus as uniquely effective, we further fractionated it to isolate the extracellular vesicles (exosomes) by Amicon tube[15], which demonstrated a highly potent inhibitory effect(~80%) as well as modulate adipogenesis genes as mRNA and protein level.
Engineering and Expression of the hisF Protein: Through proteomic analysis of the potent exosomes, we identified the hisF protein as our lead candidate. We then synthesized the hisF gene, codon-optimized it for bacterial expression, and successfully cloned it into a pET28b expression vector. This construct was used to produce recombinant hisF protein in an E. coli expression system and cell-free system.
Functional Validation and Mechanistic Studies: Finally, we purified the recombinant hisF protein and applied it to our adipocyte model. We confirmed its ability to inhibit lipid accumulation and elucidated its mechanism of action. Using qRT-PCR and Western Blotting, we verified that the hisF protein works by upregulating AMPK and downregulating the key adipogenesis markers PPARγ and C/EBPα.
Figure 5. Schematic figures of the Wet Lab Experiments
Dry Lab Experiments
To complement our wet lab findings and provide a deeper molecular understanding of our results, we conducted a series of computational analyses. Our dry lab experiments focused on investigating the structural properties of the hisF protein and its interaction with its human target, AMPK.
Comparative Structural Analysis of hisF Proteins: A key question was whether the anti-adipogenic effect was unique to the hisF protein from Lactobacillus rhamnosus. To investigate this, we used AlphaFold2 to predict and compare the three-dimensional structures of the hisF protein from three different bacteria: our lead candidate from the beneficial probiotic Lactobacillus rhamnosus, and two from common pathogens, Escherichia coli K12 and Staphylococcus aureus. By analyzing these predicted structures alongside their amino acid sequences, we aimed to identify unique structural features in the L. rhamnosus hisF protein that are not present in the others. This analysis serves to computationally justify our wet lab results, suggesting that the specific bioactivity we observed is due to a distinct protein conformation.
Modeling the hisF-AMPK Interaction: To validate the mechanism of action, we investigated the direct interaction between the bacterial hisF proteins and human AMPK. Using the advanced protein complex prediction capabilities of AlphaFold3, we modeled the formation of multimers between each of the three bacterial hisF proteins and the human AMPK complex. The goal of this simulation was to compare the predicted binding affinities and interaction interfaces. This computational docking analysis allows us to predict whether the hisF from L. rhamnosus forms a more stable and effective complex with human AMPK compared to its counterparts from other bacteria, thereby providing a molecular basis for its potent ability to activate the pathway.
Figure 6. Schematic figures of the Dry Lab Experiments