Synthetic Biology Approaches to Engineer Lactococcus lactis for Targeted Delivery of Therapeutic Lasso Peptides - ABSTRACT: Emerging strategies in synthetic biology present exciting opportunities for utilizing genetically engineered bacteria as drug delivery vehicles, offering new solutions and possibilities for drug development within the framework of precision medicine. However, there remains a gap in our knowledge regarding the development of platforms that can harness synthetic bacteria to directly synthesize and target natural product-based drugs. To address this gap, we aim to engineer a platform using genetically modified Lactococcus lactis capable of expressing biosynthetic gene clusters encoding bioactive natural products, enabling in situ drug production and targeted delivery to specific tissues. Our approach involves leveraging synthetic biology techniques to rewire the metabolic pathways of this engineered bacterium, enabling the production and release of bioactive natural products, specifically lasso peptides. This innovative proposal not only advances research in this field but also provides undergraduate students with the opportunity to develop experimental skills and foster creative thinking. We will begin by focusing on the biosynthesis of BI-32169, a compound known for its anti- diabetic and weight-control properties, by reconstructing its biosynthetic gene cluster in L. lactis. Through rational gene editing, we will modify key enzymes in the biosynthetic pathway of BI-32169 and, using structure-activity relationship (SAR)-based approaches, generate a library of bioactive analogs (BIs). These compounds will then undergo in vitro screening to identify the most potent candidates. Subsequently, we will design gene circuits to control the lysis of L. lactis within the gut environment, enabling the targeted release of these bioactive natural products. By carefully engineering these circuits, we will regulate the synthesis and release rates of the compounds to control drug concentrations. Next, we will employ an in vitro gut model to test the efficacy of the lysis circuits in releasing the bioactive compounds, optimizing the engineered bacteria for therapeutic use. Finally, we will validate the anti-diabetic efficacy of the engineered bacteria through in vivo studies in animal models. The long-term goal of this project is to establish a precise in vivo delivery system for bioactive natural products using L. lactis, while also contributing to the education of future synthetic biology talent in economically transitioning regions of the Midwest.
