Engineered Probiotic <i>Saccharomyces boulardii</i> Produces Functional Insulin With Long‐Acting Effect in Mice Model

Diabetes mellitus affects over 500 million people globally, with current insulin therapies relying on subcutaneous injections that compromise the liver's natural role in glucose regulation and suffer from poor patient compliance. Oral insulin delivery offers a physiological alternative but faces challenges, including gastrointestinal degradation and poor absorption. Here, we engineered Saccharomyces boulardii, a probiotic yeast with established gastrointestinal survival capabilities, to secrete a long-acting Insulin-Fc fusion protein (Ins/Fc). Using strong constitutive promoters (TDH3 and TEF1), we achieved stable expression in lead strains FZ030 and FZ032. The secreted Ins/Fc fusion protein demonstrated functional bioactivity in cultured cells and in diabetic mice. This represents the first successful engineering of S. boulardii to produce a functional insulin and establishes a promising platform for developing oral insulin delivery systems.