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

项目摘要/摘要 对人类微生物组的研究表明，细菌发挥着曾经被忽视但却很重要的作用 在健康和疾病中的作用。反过来，试图对微生物组重新编程以促进 健康或治疗疾病引起了越来越多的关注。使用活细菌作为药物的用处曾经是 受到它们天生行为的限制。然而，现代合成生物学工具使基因编码成为可能 功能转化为微生物，扩大了它们作为治疗药物的潜在作用。一个这样的新兴应用程序 是设计微生物，使其在肠道内充当合成工厂，使药物能够输送 来自内部的化合物。例如，被编程为提供代谢酶的活细菌疗法， 对于挽救小鼠模型中的先天代谢缺陷是有效的。迄今为止，交付的 细菌体内的治疗化合物仅限于核糖体合成的活性物质(即 酶、蛋白质、肽类激素)。尽管已知微生物的生物合成能力可以使 一系列令人眼花缭乱的小分子天然产物，现有的研究还没有利用这一点 开发以微生物为基础的治疗药物的能力。在这份提案中，我的目标是设计 益生菌大肠杆菌尼氏(ECN)生产神经药物候选前药L-4- 氯尿酸(4CK)，然后测试其在小鼠模型中的作用能力。在此过程中，我将探索 益生菌在动物宿主体内合成非天然代谢物的能力，这可能会产生新的 小分子药物持续给药的机制。 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 异源4CK生物合成途径的克隆、重构及内源调控 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 进化出生物合成酶。改良的酶变异体将被重新设计成ECN。在目标3中， 将优化菌株接种于小鼠体内，评价4CK的系统分布。这将包括 评估肠道中的产物，吸收到血液中，并传输到大脑。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，并得到了 是这些领域的专家的顾问。 。

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. .