Polyketides are a large class of natural products with interesting structural scaffolds and diverse biomedical activities. The modular nature of assembly line polyketide synthases (PKSs) has inspired ambitions to realize combinatorial biosynthesis to produce novel polyketide-based chemicals. Meanwhile, the proliferation of putative PKS gene clusters from whole genome sequencing has provided a wealth of potential building blocks. However, the broader adoption of polyketide biosynthetic engineering remains a challenging task. Designer PKS chimeras often fail to produce anticipated products in meaningful titers, reflecting an inadequate understanding into the fundamental biosynthetic logics in wild type PKSs. One hallmark of assembly line PKSs is their ability to invariably catalyze a well-defined sequence of chemical transformations with precise control on product structure and stereochemistry. Such vectorial biosynthesis requires an elaborate control mechanism to channel reactive intermediates along the catalytic cycle. In this dissertation, we seek to develop tools to analyze vectorial biosynthesis of 6-deoxyerythronolide B synthase (DEBS), a prototypical PKS, using a combination of monoclonal antibodies, small molecule activity probes and structural biology techniques. We aim to build the conceptual framework to understand the fundamental enzymology of PKSs, with an emphasis on the conformational dynamics and functional control in the context of catalysis. A major challenge to study PKSs arises from their flexible nature. A minimal functional PKS module consists of at least four distinct catalytic domains connected by flexible linkers. The full DEBS contains six such modules dispersed on three separate homodimeric polypeptide chains. The entire mega-enzyme fits in a cube of roughly 400-500 Å in dimension, where more than twenty enzymatic domains are packed. To address the protein flexibility issues, we have applied three complementary strategies. In Chapter 2, we describe development of monoclonal Fab antibody tools to study the structure and function of DEBS. By use of a fully human naive antigen-binding fragment (Fab) library displayed on a phage library, a number of high-affinity Fabs were identified against almost all the enzymatic domains central to function of DEBS. We examined these Fabs in search of potential binders to trap DEBS in catalytically relevant conformations. In Chapter 3, we describe one such Fab, 1B2, that binds to the N-terminal docking domain of DEBS Module 3. The co-crystal structure was solved to 2.1 Å resolution. Surprisingly, the X-ray crystal structure showed that this Fab locks the Module 3 in an extended conformation. Tandem size-exclusion chromatography small-angle X-ray scattering (SEC−SAXS) revealed that the same extended conformation is maintained in solution phase under conditions that preserves the maximal enzymatic activity. In vitro kinetic analysis proved that this antibody stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Taken together, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions. In Chapter 4, we report another Fab, 3A6 that recognizes the terminal thioesterase (TE) domain. Biochemical assays indicated that 3A6 binding does not inhibit enzyme turnover. The 2.45 Å co-crystal structure model revealed atomic details of the protein-protein recognition mechanism. Fab binding had minimal effects on structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bi-module. In Chapter 5, we describe our preliminary results on both negative staining and cryogenic electron microscope. Both Fab-free and Fab-bound DEBS samples have been screened. Best 3D classification result comes from 1B2-Module 2, although sample heterogeneity issues remain. In Chapter 6, we discuss the application of a small molecule activity probe and the discovery of a novel turnstile control mechanism in DEBS catalysis. In summary, this thesis attempts to expand the toolbox to investigate the detailed mechanism of PKS function and control.