Background Information

The human gut is home to trillions of microorganisms that form the gut microbiota, including bacteria, fungi, viruses, and protozoa, that coexist symbiotically with their human host. This delicate ecosystem is essential in maintaining our health, influencing everything from digestion to metabolism, immune function, and mental health (Thursby & Juge, 2017). A normal gut microbiota composition is shown below in Figure 1.

  Figure 1. Normal gut microbiota composition.

When the balance of intestinal microbial community composition is disrupted, known as gut dysbiosis, various gastrointestinal diseases might happen due to changes in metabolite production and interspecific competition. Various diseases and ailments such as Inflammatory Bowel Disease (IBD), colorectal cancer, Major Depressive Disorder (MDD), obesity, and autism are all related to gut dysbiosis (Madhogaria B. et al., 2022). Gut dysbiosis-related diseases have also become increasingly prevalent in recent years, particularly in China, as shown in Figure 2 (Yang et al., 2022; J. Li et al., 2022; N. Li et al., 2021; Zhao et al., 2021).

Figure 2. Prevalence of gut dysbiosis-related disease in China.

Current approaches to treating gut dysbiosis include taking antibiotics and fecal microbiota transplantation, while milder approaches include taking probiotics, taking prebiotics that promote probiotic growth, and changing lifestyle to attenuate inflammation (Sultan et al., 2021; Hrncir, 2022; Wei et al., 2021). These methods are not mutually exclusive and are often combined to achieve a full recovery.

Problems Faced

As shown above, gut dysbiosis is associated with multiple diseases. However, the screening for dysbiosis is insufficient, leading to failures in prevention and early identification of severe deterioration. Additionally, the treatment of gut dysbiosis is not fully developed - for now, most dysbiosis treatments favor the introduction of general probiotics such as Lactobacillus and Bifidobacterium (Bull et al., 2015), combined with drugs targeting the specific diseases. However, the gut environment is unique for each individual and very complex in composition. Thus, it is demanding for current therapies such as general antibiotics or probiotics to satisfy individual specific needs. In addition, these approaches also require periodic consultations with healthcare professionals to proscribe medicine and adjust the therapeutic schedule so that full recovery is ensured. This could be expensive, time-consuming, or inconvenient for patients. Moreover, some gut dysbiosis-related diseases, such as IBD, are not curable. Without continuous monitoring and treatment, the risk of relapse and harm accumulation is high.

The ineffectiveness of current medication and prevalent side effects emphasize the urgent need for novel, less invasive treatments. As gut dysbiosis diseases are hard to cure fully, relapses could happen frequently, while current treatments are only administered after the body exhibits initial symptoms of suffering. Thus, it is also important to develop long-term medication that could constantly monitor the gut environment, detect preliminary signs of harm, intervene, and restore balance before true pain occurs.

Thus, we envisage a probiotic platform colonizing the human gut to alleviate a wide range of gut dysbiosis-related diseases.

Our Solution

Our project aims to produce alimentary products that carry an in vivo, long-term effective, flexible, and multi-potent probiotic platform, regulating the gut microbiome to identify, prevent, and even treat harm from gut microbiome dysbiosis. Engineered metabolite biosensors strictly control the output of mediator substances, enabling the platform to detect the complex and dynamic conditions of gut microbiota dysbiosis and execute its regulatory functions.

Compared to existing treatment options, our platform has improvements. As explained, our platform aims at the regulation of the gut microbiome and its metabolites already existing in the patient rather than the introduction of outer drugs. These microbes and metabolites are normally found in the human body, so the approach is most natural and non-invasive, minimizing any side effects caused by artificial ingredients. Utilizing a living probiotic as our platform has special advantages. Living organisms are known for their ability to sensitively perceive environmental changes. Our platform leverages this capability to monitor slight changes in the gut in real time and regulate responses according to the patient's specific conditions. Through colonization, our probiotics will be able to establish a long-term presence in the human gut. This not only provides continuous surveillance to prevent relapses but also reduces intake frequency to a single administration.

1. Platform Construction

Our probiotic would be able to colonize the human intestines and exert a long-term stabilizing effect through the regulatory platform integrated into the probiotic's genome. Our probiotic will be able to detect multiple intestinal conditions timely and determine whether dysbiosis exists. If a suspected dysbiosis is detected, the probiotic would try to relieve the conditions of dysbiosis.

The detection and regulatory effect of the platform would be achieved by multiple input biosensors and output effectors. Moreover, the input sensors and output effectors on the platform can be adjusted and replaced according to specific conditions of dysbiosis.

Input sensors can misinterpret signals, for example, they may misinterpret intervals between food uptake, when the gut microbiome is not actively metabolizing as a deficiency in important metabolite production by the host's gut microbiome. Consequently, the output effectors might erroneously express enzymes for metabolite production when there is insufficient material and energy available for our probiotic to produce the metabolite, thereby increasing the burden on the metabolite. To prevent such misinterpretations and misproductions, a sensor is designed to detect the presence of chyme in the intestines. This is achieved by monitoring bile acid concentrations, which are released by the gall bladder when chyme enters the duodenum. The bile acid sensor acts as a regulatory switch for the genetic system.

In our project, we designed and tested biosensors and expressed mediators to provide usage protocols for the platform, demonstrating strict regulation and effectiveness.

Integration/Implementation

We designed two genetic systems and demonstrated that these circuits could be effective for the delivery of therapeutic molecules in the gut, both in the absence and excess of specific metabolites caused by microbiota dysbiosis.

For the absence of specific metabolites, we decided to target butyrate, an anti-inflammatory short chain fatty acid mostly produced by anaerobic bacterial fermentation. Short-chain fatty acids (SCFAs) are among the most relevant metabolites produced by gut microbiota for intestinal function, they have been shown to maintain intestinal homeostasis due to their anti-inflammatory and protective effects on the intestinal epithelium, and they participate in the regulation of multiple cellular processes. What's more, butyrate is shown to be low in patients with dysbiosis related to inflammatory bowel diease, indicating low amounts of it may be related to the diease condition. Thus, we aim at supplementing butyrate to our patient when detecting IBD related dysbiosis conditions. First, our engineered bacteria will detect low conditions of butyrate, which shows direct need for supplement. Additionally, quorum sensing molecules, specifically N-Acyl homoserine lactone (AHL) 3-oxo-C12:2, not only correlated with presence of major butyrate-producing bacteria but also serve as effective and specific indicators of IBD symptoms, with low density correlating with the presence of symptoms. Our engineered bacteria will detect low concentrations of AHL and butyrate for IBD-related dysbiosis identification. Upon detecting these signals, the bacteria will use enzymes to produce and release the butyric acid when bile acid indicates food ingestion and provides energy for butyrate production, aiming to regulate SCFA levels and alleviate the harm caused by microbiota dysbiosis in the human gut (Figure 3).

Figure 3. Genetic system targets absent specific metabolites caused by microbiota dysbiosis, dysbiosis-related IBD.

For the excess of specific metabolites, we decided to target indole-3-acetic acid (IAA). Although small quantities of IAA are beneficial to the gut environment, large concentrations of IAA could disrupt intestinal cell lineage (Wei et al., 2024). Therefore, we incorporated the repressor IacR which is sensitive to changes in IAA concentrations into the genetic circuit. Although reducing the specific IAA-producing bacteria population is preferable, directly reducing IAA concentration is feasible for our project. Therefore, we designed our bacteria to express an IAA-degrading enzyme when detecting both bile acid and excess IAA signals, as shown in Figure 4.

Figure 4. Genetic system targets the high amount of harmful metabolites.

Overall, these genetic systems may function as complementary diagnostic tools.

2. Testing Module

Input (Sensors)

Bile Acid Detection

We designed 2 systems to detect deoxycholic acid (DCA) in human gut. The first system is based on the repressor VFA0359 and the promoter pVFA0359 (Taketani et al., 2020). The second system is based on repressor BreR and the promoter pBreR. The promoters of both systems are activated at high concentration of DCA, which is required for any output in our design.

Quorum Sensing Molecule Detection

3OC12:2, as a novel AHL, has not yet been found to bind with any biological sensor. Therefore, we decided to develop a biological sensor capable of recognizing 3OC12:2.

We first attempted to create a protein that binds with 3OC12:2 using RF Diffusion and used the Blast tool to search for biological sensors with similar structures, but we did not find any similar proteins. Thus, we decided to modify the active site of an existing sensor to achieve the desired functionality.

By using batch docking, we found that AhrC, an arginine sensor, has the potential to dock with 3OC12:2. Additionally, we discovered QsrC, a protein that can dock with 3OC12, a small molecule highly similar to 3OC12:2. We transplanted the small molecule binding site of QsrC into AhrC and used molecular docking and amino acid-base docking techniques to prove its feasibility.

  Figure 5. Chemical structure of 3-oxo-C12:2 (Aguanno et al., 2020).

  Figure 6. 3D structure of 3-oxo-C12:2 drawn by Discovery Studio.

  Figure 7. 3D structure of our designed AhrC-Like protein binding of 3-oxo-C12:2.

  Figure 8. Binding of promoter with designed protein.

Short-Chain Fatty Acid Detection

Short-chain fatty acids (SCFA) such as propionate and butyrate are critical metabolites produced by the gut microbiota. Microbiome dysbiosis resulting in altered SCFA profiles is associated with certain diseases, including IBD, characterized by a reduction in butyrate concentration and active intestinal inflammation. We used molecular biosensors that quantify the absence of butyrate, utilizing logic 'NOT' gates and bacterial promoters. Promoter PpchA is derived from the part registry BBa_K4442001, and pHpdR is derived from a previous study (Wang et al., 2023). These promoters are used to sense SCFA concentrations and express the response protein Tes4 for SCFA production in amounts inversely proportional to the SCFA concentration.

Indole-3-acetic Acid Detection

We developed genetic circuits capable of sensing indole-3-acetic acid concentrations. The design contains a repressor IacR, which loses its repressing function in response to IAA. This system is used to sense IAA concentrations and induce the expression of the output proteins to degrade IAA when IAA is in excess, relieving harm from excess IAA.

Output (Production/degradation)

Short-Chain Fatty Acid Production

Upon sensing the absence of SCFA and insufficient levels of SCFA-producing bacteria, the genetic system will express SCFA for regulating, specifically butyric acid, in response.

An acyl-ACP thioesterase Tes4 (part BBa_K3838613) from Bacteroides fragilis is employed to produce butyrate. Tes4 acts on the butyryl-ACP generated by the native fatty acid biosynthesis (FASII) pathway, releasing the tetracarbon acid from ACP to result in butyric acid production.

Figure 9. Butyrate production pathway (Kallio P. et al., 2014).

Previous studies found heterologous expression of Tes4 in E. coli K27 generates low quantities of butyrate (Jing F. et al., 2011), while expressing the enzyme in E. coli BL21 produces butyrate as the main product (Kallio P. et al., 2014). Compared to alternative pathways requiring multiple enzymes, expressing this single Tes4 could achieve butyrate production, so it lowers stress on our engineered probiotic.

Indole-3-acetic Acid Degradation

Upon sensing excessive levels of IAA, the genetic system will express IAA-degrading enzymes to maintain the concentration of IAA within a reasonable value. IadCDE operons from IAA-degrading bacteria encode proteins responsible for IAA degradation (Conway et al., 2022; Scott et al., 2013). We place genes iadCDE downstream to the inducible promoter PiacR to be induced by high concentrations of IAA in the gut environment. When overexpressed, IAA is degraded with the expression of iadCDE.

3. Other Concerns

Once introduced into the human gut, the engineered bacteria will colonize and demonstrate a long-term effect in treating IBD. To prevent potential contamination from unexpected leakage, we have designed a cold-inducible kill-switch system that triggers self-destruction of the bacteria when excreted and exposed to lower temperatures. This strategy enhances the effectiveness and safety of the treatment. Additionally, we decided to use well-studied and widely-used probiotic E.coli Nissle 1917 as our chassis, ensuring our engineered bacteria itself won't pose any threat to the human body.

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