Engineering bacteria for production and delivery of the halogenated prodrug lead L-4-chlorokynurenine

用于生产和输送卤化前药先导 L-4-氯犬尿氨酸的工程细菌

• 批准号: 10607857
• 负责人: Leah Bushin
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607857
• 关键词: 7-chlorokynurenic acid; Anabolism; Animal Model; Animals; Attention; Bacteria; Behavior; Biological Assay; Biological Markers; Biomass; Biomedical Engineering; Biomedical Research; Biomedical Technology; Blood; Blood Circulation; Brain; Carbon; Catabolism; Chemicals; Circulation; Cloning; Coupled; Coupling; Development; Devices; Directed Molecular Evolution; Disease; Drug Delivery Systems; Dryness; Engineered Probiotics; Engineering; Enzymes; Escherichia coli; Evolution; Formates; Gene Deletion; Genes; Genetic; Genetic Variation; Goals; Growth; Health; Health Promotion; High Pressure Liquid Chromatography; Hormones; Human; In Situ; In Vitro; Inborn Errors of Metabolism; Invaded; Laboratories; Lead; Libraries; Measures; Medicine; Metabolic; Metabolism; Microbe; Modeling; Modernization; Modification; Multiomic Data; Mus; Mutate; Mutation; Natural Products; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Probiotics; Prodrugs; Production; Property; Proteins; Pseudomonas putida; Reporting; Ribosomes; Role; Safety; Sampling; Source; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Training; Tryptophan; Urine; Variant; Work; absorption; cancer cell; carbene; cofactor; combinatorial; design; detection limit; disorder prevention; experimental study; genome-wide; halogenation; improved; in vivo; individualized medicine; interest; marine; metabolic engineering; microbiome; microbiome research; microorganism; mouse model; multidisciplinary; mutant; non-Native; programs; sample fixation; small molecule; small molecule therapeutics; synthetic biology; tool; training opportunity

PROJECT SUMMARY/ABSTRACT Studies of the human microbiome have demonstrated that bacteria play a once overlooked, but important role in health and disease. In turn, therapeutic interventions that attempt to reprogram the microbiome to promote health or treat disease have garnered increased attention. The utility of using live bacteria as medicine was once limited by their native behavior. Modern synthetic biology tools, however, make it possible to genetically encode functions into microorganisms, expanding their potential roles as therapeutics. One such burgeoning application is to engineer microbes to function as synthetic factories within the gut, enabling the delivery of medicinal compounds from within. Live bacterial therapeutics programmed to deliver metabolic enzymes, for example, have been efficacious towards rescuing inborn errors of metabolism in mouse models. To date, the delivery of therapeutic compounds by bacteria in vivo has been limited to ribosomally-synthesized active agents (i.e. enzymes, proteins, peptidic hormones). Despite the known biosynthetic capability of microbes to make a dazzling array of small molecule natural products, there are no existing studies that have harnessed this prowess towards the development of a microbiome-based therapeutic. In this proposal, I aim to engineer the probiotic Escherichia coli Nissle (EcN) to produce the neuropharmaceutical prodrug candidate L-4- chlorokynurenine (4CK) and then test its ability to function inside a mouse model. In doing so, I will explore the capacity of probiotics to synthesize non-native metabolites inside an animal host, which could give rise to a new mechanism for the sustained delivery of small molecule drugs. In aim 1, I will genetically program EcN to synthesize 4CK and optimize production. This will require cloning and refactoring a heterologous 4CK biosynthetic pathway as well as manipulating the endogenous metabolism of the EcN chassis. In aim 2, I will use a growth-coupled selection system in Pseudomonas putida to evolve the biosynthetic enzymes. Improved enzyme variants will be re-engineered into EcN. In aim 3, the optimized strain will be administered to mice and systemic distribution of 4CK evaluated. This will include assessing production in the gut, absorption into the bloodstream and transport to the brain. This proposal is designed to provide a multidisciplinary training opportunity combining my interests in natural products, bioengineering, microbiome science, and biomedical research and is strongly supported by a diverse team of advisors who are experts in these fields. .

项目摘要/摘要 人类微生物组的研究表明，细菌曾经被忽略但很重要 在健康和疾病中的作用。反过来，试图重新编程微生物组以促进的治疗干预措施 健康或治疗疾病引起了人们的关注。将活细菌用作药物的实用性曾经 受其本地行为的限制。但是，现代合成生物学工具使得可以进行基因编码 发挥微生物的作用，扩大其作为治疗剂的潜在作用。一个迅速迅速的申请 是为了设计微生物以作为肠道内的合成工厂起作用，使药物递送 内部的化合物。实时细菌疗法已编程以提供代谢酶，例如 在挽救小鼠模型中新陈代谢的天生错误方面有效。迄今为止，交付 细菌在体内的治疗化合物仅限于核糖体合成的活性剂（即 酶，蛋白质，肽激素）。尽管微生物具有已知的生物合成能力 令人眼花and乱的小分子天然产品，没有现有的研究可以利用这一点 能够开发基于微生物组的治疗性的能力。在此提案中，我的目标是设计 益生菌大肠杆菌Nissle（ECN）产生神经药物前药L-4-- 氯脲烯氨酸（4CK），然后测试其在小鼠模型中功能的能力。这样，我将探索 益生菌在动物宿主内合成非本地代谢产物的能力，这可能引起新的 持续递送小分子药物的机制。 在AIM 1中，我将基因编程ECN合成4CK并优化生产。这将需要 克隆和重构异源4CK生物合成途径以及操纵内源性 ECN底盘的代谢。在AIM 2中，我将在Pseudomonas putida中使用增长耦合的选择系统 进化生物合成酶。改进的酶变体将被重新设计为ECN。在AIM 3中 优化的菌株将用于小鼠和评估4CK的全身分布。这将包括 评估肠道中的产生，吸收进入血液并传输到大脑。该提议是 旨在提供我对天然产品兴趣的多学科培训机会， 生物工程，微生物科学和生物医学研究，并得到了一个多元化的团队 这些领域专家的顾问。 。