Harnessing Synthetic Biology to Engineer Production and Secretion of Theanine in Saccharomyces cerevisiae

Theanine is renowned for its health-promoting properties and serves as the principal source of umami taste in green tea. Saccharomyces cerevisiae, a preferred host organism and model chassis, is often employed for the synthesis of valuable proteins and secondary metabolites. Despite the potential, achieving highly efficient theanine biosynthesis in yeast has been challenging, primarily due to the absence of comprehensive biotechnological toolkits. The rapid progress of synthetic biology motivated us to leverage standardized biological components to reconstruct the theanine biosynthetic pathway in yeast. In this study, we engineered an efficient and robust yeast strain for the biosynthesis of theanine by employing synthetic biology principles. Inducible promoter GAL1 was employed to express CsAlaDC and CsTS1, key enzymes involved in theanine biosynthesis. 276.64 mmol/L (48.19 g/L) theanine was accumulated in yeast cells when subjected to ultrasonic disruption. With the role of theanine transporter GNP1, we facilitated the secretion of theanine into the extracellular medium, obviating the need for ultrasonic disruption of yeast cells. This innovative approach demonstrated a promising step toward the industrial-scale production of theanine in yeast. Our findings offered a preliminary demonstration of the feasibility of this approach and paved the way for further advancements in the field of yeast-based theanine production.