| 6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The transparent relationship between protein sequence and enzyme function that is common to all modular PKSs makes these enzymes attractive scaffolds for the development of novel biosynthetic compounds through a process known as combinatorial biosynthesis.; Two issues that are fundamental to the development of modular PKSs as a scaffold for combinatorial biosynthesis are substrate specificity and intermodular substrate channeling. The work in this thesis investigates the substrate specificity of individual DEBS modules in the context of a diffusive-loading mechanism and in the context of a channeling mechanism.; In an in vivo experiment, a DEBS KS1 knockout system (pJRJ2/CH999) is demonstrated to be tolerant of a methoxy substituent in place of the natural methyl substituent at the α-position of an N-acetylcysteamine activated diketide, producing a novel 12-methoxy-6-deoxyerythronolide B compound.; A systematic in vitro study of the kinetic effects of stereochemical variations in diffusively-loaded diketides with individual DEBS modules purified from Escherichia coli demonstrates that all four of the tested modules manifest similar substrate specificity profiles, despite the dramatically different structures of their natural substrates. When placed in a channeling context such that the substrates are transferred from a donor ACP domain to the acceptor module, these modules also manifest similar substrate specificity profiles. However, the channeling mechanism endows a kinetic advantage of up to two orders of magnitude and a specificity advantage of up to four orders of magnitude over the diffusive mechanism.; An investigation of the relative contributions of N- and C-terminal linker interactions versus the contributions of donor ACP-acceptor KS interactions revealed that while KS domains of C-terminal modules (e.g., modules 2 and 6) are promiscuous towards unnatural upstream donor ACP domains, KS domains of N-terminal modules (e.g., modules 3 and 5) are specific for their natural upstream ACP domains. From the sum of the lessons learned through these experiments, a basic set of rules for engineering heterologous PKS assembly lines is proposed. |
