PROJECT ABSTRACT Amphotericin B is a powerful antifungal treatment however, widespread use is prevented by dose limiting toxicity. Nontoxic analogs have been discovered but their structural complexity precludes a practical chemical synthesis on industrial scale. Microbes are extraordinarily adept at producing amphotericin, as metric tons are fermented annually. Enzymes found within amphotericin’s natural biosynthetic pathway can serve as exceptionally specific and renewable biocatalysts that can overcome many common limitations encountered during its chemical synthesis. Reimagining aspects of amphotericin’s biosynthesis could therefore produce a robust and sustainable path to these nontoxic variants. This proposal aims to develop a deep understanding of several catalytic processes involved in amphotericin’s natural biosynthesis. Harnessing this knowledge, key enzymes and biosynthetic pathways will be evolved and engineered to construct a biosynthetic strategy to manufacture less toxic amphotericins. Cell-free protein synthesis provides a means to prototype these enzymes and pathways in a quantitative and high-throughput manner. Ultimately this work is expected to be an efficient platform to create new, best-in-class antifungal candidates, provide probes to interrogate structure-activity relationships dictating AmB’s potency and toxicity, and drive the continued development of enzymes as renewable, ecologically-safe and flexible biocatalysts for pharmaceutical sciences. The proposal provides ample opportunities for me to build on the knowledge from my undergraduate and graduate studies so I may continue to learn, innovate, and develop skills to be a well-rounded scientific leader. Based on the unique scientific expertise and mentoring track record of my Sponsor and Cosponsor, they are the ideal pair to provide constructive guidance in science and career development, and maximize my potential to drive this project towards success.