The MusCure project is a treatment scheme designed for inflammatory bowel disease (IBD), an intestinal autoimmune disease, using engineered bacteria for auxiliary therapy. We have designed a muscone gas molecule switch in Saccharomyces cerevisiae, which allows for the control of engineered bacteria to secrete lactic acid and inhibit the abnormal activation of immune cells, achieving the purpose of auxiliary treatment and greatly improving the treatment experience for patients. Compared to traditional treatment methods, MusCure has a lower treatment cost, minimal discomfort for patients during treatment, and more convenient treatment tools.
To verify the feasibility and social value of the MusCure project, as well as its potential future applications, we have evaluated and validated our project from three perspectives: 1. Previous literature research, 2.Experimental results, 3. Communication with relevant social stakeholders.
Although we have put in a lot of effort in the project design and experimental process, due to the limitations of the time period and technical means, there are still many deficiencies in our project design and experimental results. In response to this, we have proposed some future solutions and methods.
For the therapy system, we have conducted verification of the principles and results in three parts: muscone molecular switches, lactate secretion, and the complete system.
We hope to improve the treatment experience for IBD patients, so we have chosen gas signals as the therapeutic switch for the engineered bacterial secretion system. Drawing inspiration from the research of the Ye Haifeng team[1], we selected the muscone molecular receptor they developed and designed it to be transferred into Saccharomyces cerevisiae, to create an engineered Saccharomyces cerevisiae muscone molecular switch.
The muscone receptor is a G protein-coupled receptor derived from mouse olfactory epithelial cells, and we need to integrate the muscone receptor with the existing G protein signaling pathway in Saccharomyces cerevisiae. Referencing the research of the Benjamin M Scott team[2], we have modified the signaling pathways in Saccharomyces cerevisiae according to their operational methods and achieved the creation of a muscone molecular switch in this model organism.
For more information, please refer to Description.
Fig 1 Muscone molecular switch fluorescence signal test
A. Galactose-induced, add muscone organic solution.
B. Galactose-induced, without muscone.
We have tested the functionality of the designed muscone molecular switch in Saccharomyces cerevisiae using the GFP reporter gene intensity. Additionally, we have reduced background noise from wild-type strains by knocking out the original mating pathway receptors in Saccharomyces cerevisiae.
For more information, please refer to Therapy system.
To assess the potential for further applications of the muscone molecular switch in Saccharomyces cerevisiae, we communicated with Bluepha Company, which specializes in developing new microbial fermentation materials through synthetic biology. Bluepha Company's feedback indicated that the muscone molecular switch in our modified Saccharomyces cerevisiae could serve as an alternative to the traditional methanol promoter used in Pichia pastoris fermentation systems. Muscone offers cost-effectiveness and higher safety compared to methanol, making it an attractive option for the design of innovative fermentation processes in Saccharomyces cerevisiae.
Shortcoming: Background signal noise still exists; There are significant differences in baseline expression between different strains.
Solution: Take further measures to modify the yeast genome to eliminate background noise; Introduce the musk ketone molecular switch gene into the yeast genome to avoid the impact of plasmid cloning variations, and screen for superior yeast strains for cultivation.
We selected a small molecule drug that is relatively easy to biosynthesize and can be readily secreted to treat diseases by passing through intestinal wall cells. Through literature search, we found that Liliana M Sanmarco et. discovered a signaling pathway in which lactic acid inhibits the abnormal activation of immune cells[3]. In their study, they designed an engineered E. coli bacteria to secrete lactic acid and successfully suppressed dendritic cells and T cells in the intestinal tract. This provided a reference for our choice. We decided to introduce lactate dehydrogenase into brewing yeast, altering the anaerobic metabolism pathway to synthesize D-lactic acid for therapeutic purposes.
For more information, please refer to Description.
Fig 2 Galactose-induced D-lactic acid secretion system
We introduced a lactate dehydrogenase gene expressed by the galactose promoter into Saccharomyces cerevisiae and measured the content of D-lactic acid in the supernatant after induction. This validated that by introducing lactate dehydrogenase, we can alter the anaerobic metabolic pathway of S. cerevisiae to synthesize D-lactic acid, and the produced D-lactic acid can be secreted into the surrounding environment of the yeast.
For more information, please refer to Description.
Shortcoming: Experimental findings have shown that the nutritional composition of the yeast culture environment has a significant impact on the secretion of lactic acid; In the anaerobic environment, yeast can still produce alcohol, which may have an impact on the health of patients.
Solution:By further modifying the yeast's anaerobic metabolic pathways through genomic engineering, the impact of the nutritional composition of the culture environment on lactic acid secretion can be reduced; knockout of the alcohol dehydrogenase gene in the yeast genome.
Fig 3 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)
We have simultaneously introduced the muscone molecular switch and the downstream lactate dehydrogenase into Saccharomyces cerevisiae, constructing a complete therapeutic system within the organism. After induction with muscone, we tested the content of D-lactic acid in the culture supernatant. The experimental results demonstrate that our complete therapeutic system can achieve specific secretion of lactic acid. For further discussion on the experimental results, please refer to Therapy system.
The engineered bacteria we designed will colonize the patient's intestinal tract, achieving long-term regulated secretion for drug delivery, enhancing the stability of treatment and reducing costs. The validation of the colonization system will be conducted from two aspects: the perception of IBD signals and the colonization proteins, verifying both the principles and the results.
Out of consideration for patient safety and therapeutic efficacy, we aim to specifically colonize the engineered bacteria on the intestinal segments of the patient's lesion area. Through discussions with members of the Tsinghua 2021 team, we have selected tetrathionate as the signaling biomarker for intestinal inflammation
Referencing the research conducted by Kristina and colleagues[5], we have incorporated the TtrSR two-component system (TCS) from marine bacteria into Saccharomyces cerevisiae to function as a sensing system for detecting signals characteristic of IBD (Inflammatory Bowel Disease).
Fig 4 Fluorescence confocal microscopy imaging results (s: control group without inducer; s+: K2O6S4 added)
Fig 5 Statistical results of tetrathionate induction experiment (s: control group without inducer; s+: K2O6S4 added)
We tested the signal reporting intensity of the TtrSR two-component system introduced into Saccharomyces cerevisiae under the induction condition of tetrathionate presence using the GFP reporter gene. The results showed that the signal reporting intensity was higher than that of the control group, but not significantly. We have discussed the experimental results in detail in the Colonization system section.
To evaluate whether the signal biomarker for IBD that we used has diagnostic value, we conducted an interview with Dr. Li Yue from Peking Union Medical College Hospital. Dr. Li Yue specializes in the diagnosis and treatment of intestinal diseases and has extensive experience in the clinical diagnosis and treatment of IBD. Dr. Li Yue pointed out that, as an adjunct to treatment, using tetrathionate as a signal biomarker for IBD has certain diagnostic value, but in clinical treatment, more indicators are needed to judge the condition of the disease.
Shortcoming: The signal biomarkers cannot fully represent the condition of the disease; The binary component system is not highly sensitive in eukaryotic systems.
Solution: Using muscone as a drug secretion switch for auxiliary treatment, and employing the apoptosis system to control biological safety; by referring to the research of the team, operations such as eukaryotic codon optimization have been performed on the two-component system to make it compatible with eukaryotic systems. For details, please see the Colonization system.
By referring to the research of Wang Tianming et al.[6], we selected agglutinin-like sequence protein 3 (Als3) as the adhesion protein for Saccharomyces cerevisiae to adhere to the intestinal wall cells. This protein originates from Candida albicans and binds to epithelial E-cadherin. Due to its close phylogenetic relationship with Saccharomyces cerevisiae, we anticipate that it can produce the same adhesion effect in Saccharomyces cerevisiae as it does in Candida albicans.
Fig 6 Fluorescence confocal microscopy imaging results of attachment experiment. a, saccharomyces cerevisiae expressing Als3. b, wild- type saccharomyces cerevisiae (without Als3, control group). c, intestinal tissue section with Als3-expressing saccharomyces cerevisiae adhesion. d, intestinal tissue section with wild-type saccharomyces cerevisiae adhesion
Fig 7 Statistical results of quantification of attachment experiment (Als3: the attachment of saccharomyces cerevisiae expressing Als3; WT: the attachment of wild-type saccharomyces cerevisiae, control groups)
We determined the location of Saccharomyces cerevisiae by expressing GFP in the yeast and examined the colonization of Saccharomyces cerevisiae after co-incubation with small intestinal sections. The results confirmed that the expression of Als3 in Saccharomyces cerevisiae achieved colonization on small intestinal epithelial cells.
For more information, please refer to Colonization system.
Shortcoming: There is insufficient experimental evidence to verify the colonization effect; No complete experimental construction and testing of the colonization system have been conducted.
Solution: We will verify the expression of Als3 on the membrane of Saccharomyces cerevisiae using techniques such as Western blot; Attempt to construct and test a complete colonization system experimentally.
We discussed the design of a biosafety suicide system for intestinal environmental markers with Dr. Liu Zhihua, and Professor Liu recommended that we use bile acids as markers for the intestinal environment.
We selected bile acids as the marker for the intestinal environment, based on previous studies using the bile acid receptor FXR and its downstream BSEP promoter as the sensing system for the marker[7], and employed the Ci protein from the λ phage as an inhibitory element to suppress the expression of the toxin protein MazF.
For more information please refer to Safety.
We discussed using Saccharomyces cerevisiae as the chassis organism for our project with Dr. Liu Zhihua. Professor Liu affirmed our design of using Saccharomyces cerevisiae as the chassis organism and pointed out that Saccharomyces cerevisiae is distributed in the human intestinal environment, being a relatively harmless and safe microorganism to humans, making it very suitable as the chassis organism for our project.
We discussed the type of engineered strain of Saccharomyces cerevisiae with Dr. Li Peng. Professor Li recommended that we use the MAT alpha genotype strain, which has nutritional deficiencies in his, leu, lys, and ura. This strain can be used for transformation and screening during the project's experimental process and prevent safety threats from biological leakage.
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