This Small Business Innovation Research (SBIR) Phase I project will address the potential of synthetic biology and metabolic engineering technologies to generate microbial strains over-producing a non-caloric natural sweetener. Current production and utilization of natural sweeteners is limited due to the high cost of the cultivation and production from native plant sources. So, although natural sweeteners have been used for thousands of years, and are known for their healthy and non-caloric properties, their high production cost prevents them from directly competing with synthetic sweeteners extensively used in beverages and carbonated soft drinks. Our objective is to develop a fermentation process for biosynthetic production allowing increased adoption of low-calorie, natural sweeteners in consumer markets. Metabolic engineering approaches will be used to transfer the natural biosynthetic pathway from the plant to a bacterial host and optimize the metabolic flux for the overproduction at a commercially viable level. We anticipate that a high-productivity strain will be obtained, suitable for continued commercialization efforts. Overall, this project, if successful, will provide a new sustainable production route to the non-caloric natural sweeteners.<br/><br/>The broader impact/commercial potential of this project is the development of a microbial process for the economical and sustainable production of non-caloric natural sweetener, with a potential $3 billion global market. The use of this sweetener will improve taste profiles and expand adoption of low-calorie beverages, confectionaries, baked goods, dairy products, and so on, thus benefitting public health by reducing incidence of diabetes and other obesity-related diseases. Such benefits will translate to reduced healthcare cost both in the U.S. and globally. Additionally, this research will develop generalizable synthetic biology techniques for the high-volume production of natural products with many applications for human health and wellness.