Tunable therapeutic modulation of the gut microbiome by engineered probiotics

通过工程益生菌对肠道微生物组进行可调节的治疗调节

• 批准号: 10451749
• 负责人: Gautam Dantas
• 金额: $ 68.33万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-10 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451749
• 关键词: Affect; American; Architecture; Basic Science; Bile Acids; Biochemical; Biosensor; Cecum; Classical phenylketonuria; Colon; Communities; Complex; Data; Development; Disease; Elements; Engineered Probiotics; Engineering; Environment; Enzymes; Feedback; Gastrointestinal Diseases; Gastrointestinal tract structure; Gene Expression; Genes; Goals; Health; Human; Immune System Diseases; Immunology; In Situ; Knock-in; Libraries; Measures; Metabolic Diseases; Metagenomics; Microbe; Microbiology; Motivation; Mus; Nutrient; Pathway interactions; Patients; Phenylalanine; Phenylalanine Ammonia-Lyase; Phenylalanine Hydroxylase; Phenylketonurias; Probiotics; Production; Research; Serum; Small Intestines; Structure; Technology; Temperature; Testing; Therapeutic; Treatment Efficacy; Update; Urine; Volatile Fatty Acids; Work; colonization resistance; combinatorial; delivery vehicle; design; efficacy testing; enhancing factor; exhaustion; experimental study; gastrointestinal; gene function; gene product; genetic element; gut colonization; gut metagenome; gut microbiome; gut microbiota; host-microbe interactions; improved; in vivo; innovation; metabolic engineering; metagenome; microbial; microbial host; microbiota; microorganism; microorganism interaction; mouse model; neurotoxic; probiotic therapy; promoter; remediation; sensor; sex; synthetic biology; targeted delivery; therapeutic gene; tool

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.

摘要 工程益生菌代表着一种强大的工具，通过它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 和疾病，并提供疗法。然而，已知的益生菌由于定居而未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，受控 通过工程益生菌提供治疗药物和其他基因产品受到缺乏健壮和 用于复杂体内环境的可调合成生物学工具。建议进行这项研究的理由是 益生菌疗法的前景目前受到持久性差和缺乏强大的工程技术的限制 工具。这一建议的主要动机是了解宿主和微生物控制 益生菌整合和开发工具来设计益生菌疗法。在强劲的初步数据指引下，这 跨学科提案将追求三个具体目标：1)确定益生菌在 肠道，2)设计和优化肠道相关的生物传感器和表达电路，3)展示有效性 通过增强型益生菌定植剂体内递送苯丙酮尿症(PKU)治疗性药物。 这项建议的第一个目标是优化和识别益生菌定植的机制，测试 益生菌肠道定植通过调节其表达而增强和可调节的假说 从粪便后基因组中选择的定植因子。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植因子的详尽组合文库的益生菌 特色化的推动者。第二个目标是开发可调节基因表达的合成生物学工具 工程益生菌的控制和生物遏制，测试将传感器与 温度、pH、胆汁酸和短链脂肪酸将实现对基因表达的空间控制 沿着胃肠道。第三个目标是证明设计的益生菌底盘可以 可靠地将治疗药物输送到肠道，测试我们设计的益生菌可以稳定地 在PKU小鼠模型中应用苯丙氨酸-氨解酶(Pal2)来降低血清苯丙氨酸。 这项建议是创新的，因为我们的综合和互补的研究团队将改进 从基础科学和翻译两个层面理解益生菌疗法。拟议中的实验 它们的重要之处在于它们将1)提高我们对遗传要素和微生物相互作用的理解 管理胃肠道定植，2)产生和优化体内电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，以及3)探索替代方案的有效性， 持续给药治疗对北京大学的影响。提议的研究是有影响力的，因为它将1)开发出可靠的 益生菌定植剂，2)更新体内合成生物学应用工具箱，以及3)建立工程化 益生菌作为持续治疗输送或肠道群落结构受控调节的载体。

项目成果

Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals.

• DOI: 10.1016/j.cels.2021.10.006
• 发表时间: 2022-03-16
• 期刊: Cell systems
• 影响因子: 9.3
• 作者: Rottinghaus AG;Xi C;Amrofell MB;Yi H;Moon TS
• 通讯作者: Moon TS

Engineering microbial diagnostics and therapeutics with smart control.

具有智能控制的工程微生物诊断和治疗学。

• DOI: 10.1016/j.copbio.2020.05.006
• 发表时间: 2020-12
• 期刊: Current opinion in biotechnology
• 影响因子: 7.7
• 作者: Amrofell MB;Rottinghaus AG;Moon TS
• 通讯作者: Moon TS

Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.

• DOI: 10.1016/j.crmeth.2023.100669
• 发表时间: 2023-12-18
• 期刊: CELL REPORTS METHODS
• 作者: Amrofell, Matthew B.;Rengarajan, Sunaina;Vo, Steven T.;Tovar, Erick S. Ramirez;Lobello, Larissa;Dantas, Gautam;Moon, Tae Seok
• 通讯作者: Moon, Tae Seok

Genetically stable CRISPR-based kill switches for engineered microbes.

• DOI: 10.1038/s41467-022-28163-5
• 发表时间: 2022-02-03
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Rottinghaus AG;Ferreiro A;Fishbein SRS;Dantas G;Moon TS
• 通讯作者: Moon TS