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 对人类微生物组的研究表明，细菌在人类体内发挥着一度被忽视但重要的作用。 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 健康或治疗疾病引起了越来越多关注。使用活细菌作为药物的效用曾经是 受限于它们的原生行为。然而，现代合成生物学工具使基因编码成为可能。 功能进入微生物，扩大其作为治疗剂的潜在作用。一个这样的新兴应用 是将微生物改造成肠道内的合成工厂， 化合物从内部。例如，被编程为递送代谢酶的活细菌治疗剂， 在小鼠模型中对挽救先天性代谢缺陷有效。迄今为止， 细菌在体内产生的治疗化合物限于核糖体合成的活性剂（即， 酶、蛋白质、肽激素）。尽管已知微生物的生物合成能力， 令人眼花缭乱的小分子天然产物，目前还没有研究利用这一点， 开发基于微生物组的治疗方法的能力。在这个计划中，我的目标是 益生菌大肠杆菌Nissle（EcN），以产生神经药物前药候选物L-4-。 在一个实施方案中，使用氯犬尿氨酸（4CK），然后测试其在小鼠模型内发挥功能的能力。为此，我将探讨 益生菌在动物宿主体内合成非天然代谢物的能力，这可能会产生一种新的 用于小分子药物的持续递送的机制。 在目标1中，我将对EcN进行遗传编程以合成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。在目标2中，我将在恶臭假单胞菌中使用生长耦合选择系统 来进化生物合成酶。改进的酶变体将被重新工程化到EcN中。在目标3中， 将优化的菌株给予小鼠并评价4CK的全身分布。这将包括 评估在肠道中的产生，吸收到血液中并运输到大脑。这项建议是 旨在提供一个多学科的培训机会，结合我对天然产品的兴趣， 生物工程，微生物组科学和生物医学研究，并得到多元化团队的大力支持， 这些领域的专家顾问。 .