2023 AIChE Annual Meeting
Towards Greener Production of Complex Molecules: Enhancing Triterpenoid Production in Yeast Via PAH1 Gene Mutation
Triterpenoids, one of the largest classes of phytochemicals, are valuable polycyclic molecules that can be applied as pesticides, pharmaceuticals, and more. Typically, the main production mode for triterpenoids is direct extraction from plants, however yeast cell factories may prove to be a more cost-effective alternative. The objective of this project is to optimize a strain of S. cerevisiae for triterpenoid production through alteration of the PAH1 gene. PAH1 codes for the enzyme phosphatidic acid phosphatase, which converts phosphatidic acid to diacylglycerides within a cell membrane. Disrupting synthesis of the PAH1 enzyme has been shown to alter the phospholipid composition of the cell membrane, increasing its permeability and facilitating the export of triterpenoids into growth media to prevent the toxic buildup of triterpenoids inside the cell. The CRISPR-Cas9 gene editing system was used to target and mutate the PAH1 gene, first by using a knock-out method (non-homologous end-joining). Resulting mutant strains of S. cerevisiae were isolated and sequenced, which revealed varying frameshift mutations. The location of base pair deletions was consistent across strains, though the number of missing base pairs differed. The chemical profile of both mutant and wild type strains was analyzed via GC-MS to determine whether the mutant strains produce higher levels of triterpenoids than the wild type. To create a genetic disruption more likely to cause a phenotype and to optimize the yeast engineering process, the same CRISPR-Cas9 system is being used to perform a gene knock-in (homology-directed repair) with donor DNA containing numerous stop codons. Overall, we demonstrate that gene editing with CRISPR-Cas9 is effective in S. cerevisiae, paving the way for further genetic modification and the continued optimization of triterpenoid production with yeast cell factories.