(153f) Improving Therapeutic Protein Secretion in Probiotic Yeast Saccharomyces Boulardii using a Multifactorial Engineering Approach.

Recent advancements in synthetic biology enabled emergence of engineered live biotherapeutic products (LBPs). Engineered probiotics, as a class of engineered LBPs, are designer microbial therapies to target gastrointestinal disorders by delivering therapeutic molecule at the disease site [1]. Yeast as a eukaryotic cell factory poses several biomanufacturing advantages over bacteria for in situ drug delivery applications, such as ability to secrete high titers of protein and post-translational modification machinery. Particularly, probiotic yeast Saccharomyces boulardii (Sb) is a promising engineering chassis for next-generation probiotics applications such as in situ biomanufacturing of small molecules and therapeutic proteins in the gut [2,3].

In this work, we investigated means to improve recombinant protein secretion in Sb. We used 2 anti-Clostridioides difficile peptides and 4 anti-Clostridioides difficile nanobodies as model proteins. To validate and quantify secreted therapeutic peptide in the supernatant, we used SDS-PAGE and ELISA. After validating the secretion of recombinant proteins, we harnessed 3 strategies to tune protein secretion levels in engineered Sb. First, we investigated the effect of expression vector on the protein concentration in the supernatant. We compared genome-integrated vectors and plasmidic vectors with low- and high-copy origins. Second, we investigated the effect of secretion signals on therapeutic protein concentration. We screened 6 signal sequences, 3 native signals and 3 engineered signals. Thirdly, we engineered the major steps in the secretory pathway in Sb to improve the therapeutic protein secretion. We created a knock-out library of 17 Sb strains via CRISPR-Cas9 genome editing and screened for protein production and growth using microbioreactors. Lastly, we investigated the in vivo colonization and excretion profiles of engineered Sb strains in mice models. Through this engineering effort, we identified Sb strains with over 10-fold higher protein secretion levels than wild-type Sb, and over 4 orders of magnitude higher secretion levels than probiotic bacteria strains. Overall, this work lays the foundation for tuning recombinant protein secretion in Sb and establishing Sb as in-gut therapeutic biosynthesis platform.