The yeast Saccharomyces cerevisiae has been an important host for the biosynthesis of valuable products in a diverse range of industrial sectors, ranging from energy to food and beverage to biopharmaceutical. With the advent of synthetic biology, several important molecular tools have been developed for engineering the yeast to be a more efficient microbial cell factory and to elucidate the vast interacting gene networks for better fundamental understanding. In this research, we have developed, validated, and applied two novel synthetic biology tools for S. cerevisiae; these can be used for the improvement of product synthesis and to examine yeast cellular processes. The first tool utilizes CRISPR-Cas9, a potent gene editing technique, to generate a medium-sized random combinatorial gene knockout yeast library. This was used to quickly and efficiently identify positive combinations of pathway and regulatory genes related to the accumulation and utilization of the acetyl-CoA and malonyl-CoA pool within the yeast cytosol. The generation of combinatorial gene knockout yeast libraries via CRISPR-Cas9 and the yeast culture conditions were first optimized to remove any bias and allow reproducibility, respectively. Using our new method, we identified yeast strains exhibiting up to 3-fold differences in specific titer of the polyketide triacetic acid lactone (TAL). This rapid low-cost approach allows generation and screening of yeasts cells containing random combinations of gene knockout with minimal bias, eliminating the need for the tedious process of sequentially knocking out potential gene targets. The second synthetic biology tool emulates natural enzyme colocalization as a strategy to increase the biosynthesis of heterologous products in yeast. We have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6-mediated RNA-guided protein assembly within the yeast cytosol. This scaffold was used to bring together a split luciferase in S. cerevisiae, resulting in a 3.6- to 20-fold increase in luminescence signal compared to the controls. Temporal control of the scaffold was also successfully demonstrated. Next, the scaffold was successfully applied to increase the amount of deoxyviolacein (desired) by 2-fold relative to prodeoxyviolacin (undesired) in a partial violacein pathway. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in up to 1.9-fold increase in luminescence signal. Furthermore, this Cas6-mediated RNA scaffold has shown promise to colocalize the enzymes acetyl-CoA carboxylase and 2-pyrone synthase into a metabolon to improve synthesis of TAL, and for forming functional degradation complexes for targeted ubiquitination and protein degradation. These are the first studies that utilize RNA as synthetic protein scaffold within the yeast cytosol. The flexibility of the design suggest that this strategy can be used to create metabolons and for other applications in a wide range of recombinant hosts of interest.