Discovery, Expression,\nand <i>In Silico</i> Safety Evaluation of Honey Truffle\nSweetener, a Sweet Protein Derived\nfrom Mattirolomyces terfezioides and\nProduced by Heterologous Expression in Komagataella\nphaffii

Honey truffle sweetener (HTS), a\n121 amino acid protein is identified\nas a high-intensity sweetener found naturally occurring in the Hungarian\nSweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely\nsweet, but the truffle is difficult to cultivate; therefore, the protein\nwas systematically characterized, and the gene coding for the protein\nwas expressed in a commonly used host yeast Komagataella\nphaffii. The heterologously expressed protein maintained\nthe structural characteristics and sweet taste of the truffle. Preliminary\nsafety evaluations for use as a food ingredient were performed on\nthe protein including digestibility and <i>in silico</i> approaches for predicting the allergenicity and toxicity of the\nprotein. HTS is predicted to be nonallergenic, nontoxic, and readily\ndigestible. This protein is readily produced by precision fermentation\nof the host yeast, making it a potential replacement for both added\nsugars and small molecule high-intensity sweeteners in food.