FERMENTATION OF UNDESIRABLE OLIGOSACCHARIDES BY DESIGNER BAKER�S YEAST TO ALLEVIATE FUNCTIONAL BOWEL DISORDERS

Our long-term goal is to develop a new class of engineered yeast strains for the targeted removal of FODMAP in the dough fermentation process. Remarkably little is known about how yeast-secreted enzymes interact with FODMAPs. Therefore, our project goalis to establish a rational framework for engineering baker's yeast to reduce FODMAP levels during dough fermentation. Our central hypothesis is that baker's yeast designed to efficiently secret FODMAP-specific enzymes will allow complete degradation of FODMAPs via dough fermentation, resulting in health-beneficial effects for people suffering from gastrointestinal disorders. This hypothesis was formulated based on previous studies showing that variability in yeast invertase activity determined the extent of fructan hydrolysis during dough fermentation. We propose to achieve our project goal by pursuing the following three specific objectives.Objective 1. Engineer enzyme secretion pathways in baker's yeast. Our working hypothesis is that improved enzyme secretion by baker's yeast will enhance FODMAP degradation and CO2 formation, reducing total fermentation time. We will engineer a set of Saccharomycescerevisiae capable of efficiently secreting enzymes. The strain will be subjected to UV mutagenesis to create a strain library for screening.Objective 2. Engineer S. cerevisiae invertase activity and specificity toward fructan. Hydrolysis of target the FODMAP, fructan, is catalyzed by yeast invertase, but baker's yeast invertase shows inherent inefficiency in fructan degradation. We hypothesize that efficient and rapid degradation of fructan depends on invertase activity and specificity. We will combine directed evolution with a colony size-based screening method to identify direct and indirect factors that elicit high levels of invertase activity and specificity toward fructan.Objective 3. Develop a baker's yeast platform for expressing recombinant fructanase. An alternative fructan-degrading enzyme from other yeast strains has the potential to surpass the performance of baker's yeast invertase. Our working hypothesis is that a dual enzyme expression system, including S. cerevisiae invertase and fructanases from other GRAS microorganisms such as Kluyveromyces marxianus and Aspergillus niger, will leverage continued advances in strain performance related to fructan hydrolysis. We propose to introduce a recombinant fructanase system, inducing a synergistic effect of multiple enzymes for FODMAP breakdown.