Redirecting metabolic flux requires the knockout of enzymes and regulatory factors limiting product formation. However, gene knockouts do not allow for the modulation of essential genes accounting for up to 20% of genes within a cell. One method to access these genetic targets and maximize product flux is through limiting the expression of essential genes using a gene knockdown approach. In this research project, fine-tuned genetic control using a synthetic low-expression promoter library will be employed to knockdown the expression of essential genes limiting the production of carotenoids and lipids in two distinct yeast systems. To accomplish this, a functional library of low-expression promoters will be developed and characterized through the use of a competitive titration-based genetic screen. These promoters will be integrated into the genome using a promoter replacement cassette to reduce the expression of essential genes identified through a modified stoichiometric modeling approach. Finally, the research team will evaluate the tradeoff between improved product formation and reduced growth. This approach allows for a better understanding of both metabolic pathways and essential gene function while providing a novel approach to manipulating pathways.
