In the future, we plan to characterize our system by growing liquid cultures and assaying samples for ascorbic acid at incremental time intervals to establish rate constants as well as quantify how much vitamin C (ascorbic acid) our system can produce. We also hope to decisively quantify how much beta-carotene our VitaBread contains in order to assess the viability of the applications that we envision for our VitaYeast.
Once we have Vitamin C over time curves we will run a comparative study on how yeast producing Vitamin C grows on YPD plates (a lab standard) versus how it grows on our desired substrate, bread. To test its growth on bread we have created plates of dough like media.
To optimize the production of ascorbic acid on the new substrate we will apply a strategy of directed evolution. We will make a combinatorial library of expression cassettes using golden gate assembly for every synthesized orf in the Vitamin C pathway. This will be made using promoters and terminators of varying strengths from across the genome. Once constructed, this library will be plated on dough media plate with a calculated amount of peroxide in the agar. The presence of the peroxide will apply oxidative stress to the cells and as Vitamin C confers oxidative stress resistance this behaves as a selection for the cells that producing more Vitamin C. By adjusting the amount of peroxide on the plate we can create a threshold of ascorbic acid production below which all the cells will die. To establish which one of the library members that passed through the selection is the best, we will perform a quantitative screen on the survivors. This will be done by growing up the cells in liquid culture and then extracting the ascorbic acid and spectrophotomertically determining its concentration.
As the quantitative measurements proved successful for our strains of yeast producing β-carotene, one of the future directions of our project is to perform the same quantitative analysis of product formation and gene expression for our Vitamin C producing strain of yeast. In addition, we plan to perform quantitative analysis of kinetic rates of our enzymes. As we have already transformed the tagged enzymes in the β-carotene pathway, purification and kinetics assays will be performed within the next month. In addition, the same methodology will be applied for the Vitamin C enzymes; therefore this goal is very realistic. These measurements will produce novel kinetic rates for these enzymes in Xanthophyllomyces Dendrorhous, which currently are not in literature. After transforming our final strain with both the β-carotene and Vitamin C genes, we will perform the same enzyme kinetics and product formation, allowing us to most accurately optimize our system and produce the most effective strain for the consumer.