In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.
In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.
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DOI：
10.14440/jbm.2021.347
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Soares JW
中科院分区：
文献类型：
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作者：
Arcidiacono S;Breedon AME;Goodson MS;Doherty LA;Lyon W;Jimenez G;Pantoja-Feliciano IG;Soares JW
体外发酵系统为了解多微生物群落内复杂的代谢动力学提供了重要机会，特别是与人类肠道微生物群相关的微生物群落。体外肠道模型具有广泛的实验容量，允许快速评估多个参数，生成知识，为后续体内研究的设计提供信息。在这里，我们的方法描述了一个体外发酵试验台，以提供对工程益生菌电路设计功能的生理相关评估。通常，工程益生菌在原始、单一或联合培养条件下进行评估，并直接过渡到动物或人类研究，当被引入复杂的肠道群落时，通常会导致预期功能的丧失。我们的方法包括一个系统的工作流程，包括发酵、分子和功能表征以及统计分析，以验证工程益生菌的持久性、质粒稳定性和报告程序响应。为了演示这一工作流程，利用简化的人类肠道微生物共生体的多菌群落来研究益生菌大肠杆菌Nissle 1917，该益生菌被设计为生产荧光报告蛋白。随着复杂性的增加，共生体被组合在一起，以产生一个基于养分利用的模拟群落。该方法评估了工程益生菌在竞争生长环境中的持久性、报告产品和功能、工程对生物生长的影响以及对共生成分的影响。体外试验台代表了设计-建造-测试-学习范式中的一个新元素，为电路重新设计提供生理相关的反馈，并为工程益生菌向更高保真度动物或人类研究的过渡提供实验验证。
In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, mono- or co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic’s persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.