Metabolic Flux Balance Analysis for Prediction of Targeted Genetic Modifications for the Increased Production of Beta-Carotene by Recombinant S. cerevisiae

Carotenoids are an important group of molecules found in many biological systems. They are a precursor to vitamin A and are used in vitamins and supplements, and in food dyes. Carotenoids are produced in plants in low quantities, and chemical synthesis is not a viable option, so overproducing this molecule in organisms such as yeast is the most economical choice in an increasing global market. Previous work has been done by Reyes et al. [Metabolic Engineering, 21, 26-33 (2014)] to engineer a strain of Saccharomyces cerevisiae that can over-produce carotenoids. It is now desired to increase the production of carotenoids in order for this process to become cost-competitive. This work first examines the substrate utilization and carotenoid production through the use of a modified metabolic flux model of S. cerevisiae. In addition, this model was combined with an extracellular kinetic model, leading to better understanding and prediction of carotenoid production. The metabolic flux model was also used to identify genes, including genes involved in squalene and pyruvate pathways, whose absence could lead to higher carotenoid productivity. Several genes were disrupted using a form of the CRISPR/Cas-9 system developed by Bao et al. [ACS Synthetic Biology, 4, 585-594 (2015)]. This system uses a guided Cas9 nuclease protein to create a double-stranded break in the DNA at targeted gene locations. Subsequent homologous recombination creates frame-shift mutations that render the targeted gene(s) non-functioning. These targeted gene knockouts in the engineered S. cerevisiae strain impact both the growth and productivity of carotenoids.