Natural products have a long history of use in traditional and modern medicine due to their inherent bioactivity. Some medicinal activities include antibiotic, antifungal, anticancer, antiviral, antihypercholestrolemic, and immunosuppressant. One of the largest classes of bioactive natural products are polyketides, produced by polyketide synthases (PKS). PKS are closely related to fatty acid synthases (FAS), sharing core biosynthetic logic that uses simple precursor molecules, but there are key differences in PKS that lead to the incredible diversity of structure observed in nature. Polyketide biosynthesis can be grouped into initiation, extension, reduction, aromatization and cyclization, and tailoring steps. Changes at each step have the potential to produce many variations in structure and this has sparked incredible interest in studying PKS chemistry to engineer these systems to produce novel medicinal compounds. Early engineering attempts mixed PKS components which could produce new polyketides but suffered from reduced yields and low fidelity. Furthermore, it was common for some combinations to not work at all, indicating that more investigation is necessary to improve engineering success. Three major challenges are addressed in this dissertation span natural product drug discovery and PKS biosynthesis. The first challenge is COVID-19’s ongoing threat to human health. While COVID-19 does not have the case fatality rate of its predecessors, SARS and MERS, its ability to spread quickly led to global shutdowns. Currently, several effective vaccines are available but small molecule drugs, while maintaining effectiveness across multiple viral strains, are limited in scope. Therefore, there is a pressing need to find new compounds that effectively inhibit SARS-CoV-2 infection. The intrinsic bioactivity displayed by natural products makes them an excellent source for new drugs therefore, molecular docking was employed (chapter 2) to identify promising compounds for further study from a library of over 200. The second two challenges are from polyketide biosynthesis at the initiation and reduction steps. Initiation begins polyketide biosynthesis, and one or more enzymes can be involved to select a particular chemical moiety as a polyketide’s first building block. The most common enzyme is the priming ketosynthase, but the molecular factors that determine its selectivity for the first building block remain unclear. At the reduction step, the substrate is a long, poly-β-ketone chain that is prone to spontaneous cyclization. This precludes structurally characterizing the ketoreductase responsible for regiospecific reduction at the ninth carbon and C7-C12 first ring cyclization with the native substrate. To elucidate initiation selectivity, three structures of the priming ketosynthase ZhuH from the R1128 system were solved with different acyl groups bound to the active site cysteine. How these acyl chains interact with active site residues elucidated the role of ZhuH in selecting its building block (chapter 3). Substrate reactivity at the reduction step was overcome by co-crystallizing the actinorhodin ketoreductase with two generations of substrate mimic. The resulting structures provided the first insight into how the substrate is positioned in the active site (chapter 4).