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

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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. .