Tunable therapeutic modulation of the gut microbiome by engineered probiotics

摘要 工程益生菌代表了一种强大的工具，可以将基因功能和途径“敲入”到 肠道微生物组，改变肠道微生物组的结构，以测试关于群落结构的假设 and disease疾病，and to deliver交付therapeutic治疗.然而，已知的益生菌由于定植而不能在肠道中持续存在 肠道微生物群的耐药性，限制了它们作为研究或治疗工具的价值。此外，控制 通过工程益生菌递送治疗剂和其他基因产品受到缺乏强大且 用于复杂体内环境的可调合成生物学工具。拟议研究的基本原理是 益生菌疗法的前景目前受到持久性差和缺乏强大工程的限制 工具.这项建议的中心动机是了解宿主和微生物的机制， 益生菌整合和开发工具，工程益生菌疗法。在强大的初步数据指导下， 跨学科建议将追求三个具体目标：1）确定益生菌定植的决定因素， 肠道，2）设计和优化肠道相关的生物传感器和表达电路，以及3）证明功效 通过增强的益生菌定殖剂体内递送苯丙酮尿症（PKU）治疗剂。 该提案的第一个目的是优化和鉴定益生菌定植的机制，测试益生菌定植的可能性。 假设益生菌肠道定植通过调节 定植因子选自粪便宏基因组。特别是，我们将选择持久的殖民者 来自表达由体内驱动的定殖因子的详尽组合文库的益生菌 特征启动子。第二个目标是开发可调基因表达的合成生物学工具 控制和生物遏制工程益生菌，测试的假设，结合传感器， 温度、pH、胆汁酸和短链脂肪酸将能够对基因表达进行空间控制 沿着胃肠道。第三个目的是证明工程益生菌底盘可以 可靠地将治疗药物输送到肠道，测试我们的工程益生菌可以稳定地 在PKU的鼠模型中递送苯丙氨酸-氨裂解酶（Pal 2）以降低血清苯丙氨酸。 这项建议是创新的，因为我们的综合和互补的研究团队将提高 在基础科学和转化水平上理解益生菌疗法。拟议的实验 重要的是，它们将1）提高我们对遗传因素和微生物相互作用的理解 控制胃肠道定植，2）生成和优化用于体内回路控制的合成生物学工具 这将是模块化的，并广泛适用于益生菌工程，和3）探索替代品的功效， PKU的持续输送治疗剂。拟议的研究是有影响力的，因为它将1）发展可靠的 益生菌定植者，2）更新合成生物学体内应用的工具箱，以及3）建立工程化的 益生菌作为载体用于持续治疗递送或肠道群落结构的受控调节。

ABSTRACT Engineered probiotics represent a powerful tool with which to ‘knock-in’ gene functions and pathways into the gut microbiome, alter the structure of the gut microbiome to test hypotheses regarding community architecture and disease, and to deliver therapeutics. However, known probiotics fail to persist in the gut due to colonization resistance by the gut microbiota, limiting their value as either research or therapeutic tools. Further, controlled delivery of therapeutics and other gene products by engineered probiotics is limited by a lack of robust and tunable synthetic biology tools for the complex in vivo environment. The rationale for the proposed research is that the promise of probiotic therapies is currently limited by poor persistence and a lack of robust engineering tools. The central motivation for this proposal is to understand the host and microbial mechanisms governing probiotic integration and develop tools to engineer probiotic therapies. Guided by strong preliminary data, this interdisciplinary proposal will pursue three specific aims: to 1) identify determinants of probiotic colonization in the gut, 2) design and optimize gut-relevant biosensor and expression circuits, and 3) demonstrate the efficacy of in vivo delivery of a phenylketonuria (PKU) therapeutic by an enhanced probiotic colonizer. The first aim of this proposal is to optimize and identify mechanisms of probiotic colonization, testing the hypothesis that probiotic gut colonization is enhanced and tunable by modulating expression of colonization factors selected from fecal metagenomes. In particular, we will select for durable colonizers from probiotics expressing an exhaustive combinatorial library of colonization factors driven by in vivo characterized promoters. The second aim is to develop synthetic biology tools for tunable gene expression control and biocontainment of engineered probiotics, testing the hypothesis that combining sensors for temperature, pH, bile acids, and short chain fatty acids will enable spatial control over gene expression along the gastrointestinal tract. The third aim is to demonstrate that the engineered probiotic chassis can reliably deliver therapeutics to the gut, testing the hypothesis that our engineered probiotic can stably deliver phenylalanine-ammonia lyase (Pal2) in a murine model of PKU to decrease serum phenylalanine. This proposal is innovative because our integrated and complementary research team will improve understanding of probiotic therapies at both basic science and translational levels. The proposed experiments are significant in that they will 1) improve our understanding of the genetic elements and microbial interactions governing gastrointestinal colonization, 2) generate and optimize synthetic biology tools for in vivo circuit control that will be modular and widely applicable to probiotic engineering, and 3) explore the efficacy of an alternative, continual-delivery therapeutic for PKU. The proposed research is impactful because it will 1) develop reliable probiotic colonizers, 2) update the toolbox for synthetic biology in vivo applications, and 3) establish engineered probiotics as vehicles for sustained therapeutic delivery or controlled modulation of gut community architectures.