To date, at least twenty enzymes in E. coli have been identified to functionally interact with the acyl carrier protein (ACP), a small, four-helix bundle that shuttles thioester-linked pathway intermediates to the active site of partner enzyme (PE). About half of these PEs are fatty acid synthases (FASs) that catalyze the primary metabolic pathway, fatty acid biosynthesis (FAB), to synthesize saturated and mono-unsaturated fatty acids. FAB goes through iterative cycles that condense 2-carbon unit at a time onto the growing acyl chain. During this process, FASs ensure product fidelity by applying accurate substrate recognition and maintain catalytic efficiency by forming productive protein-protein interactions (PPIs) with ACP. The modular biosynthetic logic of FASs, and the evolutionarily related polyketide synthases (PKSs), makes them attractive metabolic engineering targets, but a thorough understanding of the underlying enzymology is needed to better achieve the goal. Here, we utilize the PE-ACP crosslinking methodology to investigate the enzymology of three ACP-dependent synthases that catalyze carbon-carbon bond formation through decarboxylative condensation. These synthases are elongating ketosynthases (KSs), FabF and FabB, from FASs, and the 8-amino-7-oxononanoate synthase, BioF, from the biotin biosynthesis pathway. The first chapter provides an extensive review on the FASs and PKSs, with a focus on PPIs and the biochemical tools that are used to study these enzymes. The well-developed biochemical techniques, especially the mechanism-based crosslinking method, has allowed us to rapidly leverage our understanding of KS enzymology in the past three years (2019-2021), and this leads to the second chapter that provides a comprehensive overview on mechanisms governed by KSs. In the third chapter, a recently discovered double-loop gating mechanism was closely examined by mutational study of FabF. We also reported two FabF-ACP structures that capture the gate in different conformations. Although highly similar to FabF in function, FabB has a different substrate preference and is essential to unsaturated fatty acid (UFA) synthesis. In chapter four, crosslinkers that mimic specific stages of catalysis were used to probe the substrate specificity of FabB. The two reported FabB-ACP complex structures also delineate gating mechanisms utilized by the enzyme. Finally, in the fifth chapter, we turn our focus to a PLP-dependent (Pyridoxal 5’-Phosphate) biotin synthase, BioF, that catalyzes the condensation between pimeloyl-ACP and L-alanine. We developed a SuFEx-based (Sulfur (VI) Fluoride Exchange) crosslinker that successfully led to the BioF-ACP complex structure. From structural analysis and mutational study, we identified a BioF-ACP interface that was different from what in silico docking suggested.