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

项目摘要 工程化的微生物代表了一个有前途的平台，用于控制微生物代谢， 肠道微生物群对治疗效果的影响。虽然已经成功地改造了菌株， 毒性代谢物的浓度或产生治疗性代谢物，能够控制 在狭窄的窗口内的代谢物尚未开发。这种“智能益生菌”，能够动态地 对环境作出反应，并根据当地浓度生产或消耗一种化合物， 特别适用于稳定在宿主健康中起浓度依赖性作用的代谢物， 疾病例如，溃疡性结肠炎和克罗恩病与微生物产生的氢有关 硫化物（H2S），越来越多的共识是，低水平的这种分子具有抗炎特性， 支持健康上皮，而高浓度的H2S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H2S浓度在空间上变化， 并且暂时地遍及粘膜，在小电流的情况下不可能将H2S控制在紧密的范围内。 分子硫化物供体，其释放硫化物而不管局部浓度。我们提出了一种新的合成 基于生物学的原位控制微生物代谢物的方法，其中工程微生物使用 转录因子响应于感兴趣的代谢物，以动态平衡代谢产物的表达。 代谢物的生产和消耗途径。这将产生稳定的可滴定浓度 以类似于恒温器的方式。在本提案中，我们将通过开发 工程菌株E.大肠杆菌Nissle动态控制H2S的水平在原位，结合数学 将建模和人体器官芯片平台纳入设计-构建-测试周期，以实现稳健和稳定的操作 在复杂的肠道环境中。如果成功的话，这项研究将为一部小说建立设计规则。 合成生物学控制策略适用于许多具有浓度依赖性作用肠道代谢物， 疾病，识别和减轻影响工程菌株性能的宿主因素， 合成益生菌的可翻译性。

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.