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Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides , an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii . The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae .