Engineered Probiotics for Closed-Loop Control of Disease-Associated Gut Metabolites in Gut-On-Chip Models

项目总结 工程共生微生物代表着控制微生物代谢的一个有前途的平台 肠道微生物群对治疗结果的影响。虽然已经成功地对菌株进行了改造，以减少 有毒代谢物的浓度或产生治疗性代谢物的菌株，能够控制 窄窗口内的代谢物还没有被开发出来。这样的“智能益生菌”，能够动态 对环境做出反应，并根据当地浓度生产或消费化合物，将 对于稳定在宿主健康中起浓度依赖作用的代谢物特别有用 疾病。例如，溃疡性结肠炎和克罗恩病与微生物产生的氢气有关 硫化物(硫化氢)，越来越多的人一致认为，低水平的这种分子具有抗炎特性和 支持健康的上皮，而高浓度的硫化氢具有遗传毒性，抑制线粒体功能 和丁酸氧化，并潜在地削弱粘膜屏障。考虑到硫化氢浓度在空间上的变化 从时间上讲，在整个粘膜中，用小电流控制H_2S是不可能的。 分子硫化物供体，无论局部浓度如何，都会释放硫化物。我们提出了一种新的合成方法 基于生物学的方法在原位控制微生物代谢物，其中工程微生物使用 转录因子反应感兴趣的代谢物以动态平衡代谢产物的表达 代谢物的生产和消费途径。这将产生稳定的、可滴定的浓度 以类似于恒温器的方式。在这份提案中，我们将通过开发 工程菌株E.ColiNissle在现场动态控制硫化氢水平，结合数学 建模和人体器官芯片平台进入设计-建造-测试周期，实现稳健稳定运行 在复杂的肠道环境中。如果成功，这项拟议的研究将为一部小说建立设计规则 一种适用于多种具有浓度依赖作用的肠道代谢产物的合成生物控制策略 疾病，识别和减轻影响工程菌株性能的宿主因素，并促进更多 合成益生菌的可译性。

PROJECT SUMMARY Engineered commensal microbes represent a promising platform for controlling microbial metabolism in the gut microbiota for therapeutic outcomes. While strains have been successfully engineered to either reduce the concentration of a toxic metabolite or produce a therapeutic one, strains capable of controlling the level of a metabolite within a narrow window have not been developed. Such ‘smart probiotics’, able to dynamically respond to the environment and either produce or consume a compound based on the local concentration, would be particularly useful for stabilizing metabolites which play a concentration-dependent role in host health and disease. For example, ulcerative colitis and Crohn’s disease have been linked to microbially produced hydrogen sulfide (H2S), with a growing consensus that low levels of this molecule have anti-inflammatory properties and support a healthy epithelium, whereas high concentrations of H2S are genotoxic, inhibit mitochondrial function and butyrate oxidation, and potentially weaken the mucosal barrier. Given that H2S concentration varies spatially and temporally throughout the mucosa, controlling H2S within a tight range is not possible with current small- molecule sulfide donors, which release sulfide regardless of local concentration. We propose a new synthetic biology-based approach to controlling microbial metabolites in situ, in which the engineered microbe uses a transcription factor responsive to the metabolite of interest to dynamically balance the expression of metabolic pathways for production and consumption of the metabolite. This will produce a stable, titratable concentration in a manner analogous to a thermostat. In this proposal, we will demonstrate this technology by developing engineered strains of E. coli Nissle to dynamically control the level of H2S in situ, incorporating mathematical modeling and a human organ-chip platform into the design-built-test cycle to achieve robust and stable operation in the complex gut environment. If successful, the proposed research will establish the design rules for a novel synthetic biology control strategy applicable to many gut metabolites with concentration-dependent roles in disease, identify and mitigate host factors that impact engineered strain performance, and facilitate greater translatability of synthetic probiotics.