2023 37 B2

Optimizing Biochemical Production in S. cerevisiae

By: John Gonzales

Department: Biology

Faculty Advisor: Dr. Mark Chan

Saccharomyces cerevisiae (also known as brewer's yeast or baker's yeast) is one of the most widely used experimental organisms in biotechnology. We believe that organelles may be used and engineered as bioreactors - vessels that carry out biochemical reactions. In this presentation, we test the hypothesis that the size of organelles like the yeast vacuole impacts their capacity to store potential products of interest. To efficiently engineer yeast vacuoles as bioreactors to produce compounds of interest in larger scales, it is vital that we develop a firm understanding of how vacuolar size affects compound accumulation. We do this utilizing well-described mutants of S. cerevisiae with a non-functional allele of ADE2. This loss-of-function mutation leads to the accumulation of aminoimidazole ribonucleotide (AIR), an intermediate in adenine synthesis and our compound of interest, in the vacuoles of yeast cells when they are grown in medium that lacks adenine. AIR naturally fluoresces red when exposed to oxygen, which allows us to measure its accumulation in vacuoles using fluorescence microscopy. By utilizing spinning disk confocal microscopy, AIR accumulation was measured in ade2 mutants whose vacuole membranes were also tagged with green fluorescent protein (VPH1-GFP). Our initial results indicate a positive correlation between vacuolar size and the amount of AIR accumulated. Future experiments will extend on these results, and I will be utilizing an optogenetic system in order to increase vacuole size, which in theory would lead to an increase in AIR production.