Polyketide synthases produce a remarkable number of diverse products. Many medicines have been produced either directly from polyketide products found in nature, or with limited modification via organic semisynthesis. However, we remain in the early days of engineering the biosynthesis of polyketide synthases. Despite being an active area of research for over 20 years, no commercial application of an engineered polyketide synthase exists. Rapidly advancing technologies like next generation sequencing, DNA synthesis, liquid handling automation and mass spectrometry give us tools to do things faster, and in new ways. In the work presented in this doctoral dissertation I will describe how I have applied some of these new tools to advance our understanding of polyketide synthase biochemistry, as well develop catalysts for new small molecules using engineered polyketide synthases.
Chapter 1 begins with a discussion of the current state of the art and challenges associated with engineering these enzymes as well as offering opinions about routes forward to narrow the considerable gap between the promise and reality of engineered PKSs.
Chapter 2 presents my work applying mass spectrometry to study the formolase enzyme, which is part of an engineered carbon fixation pathway. This work further characterized the product profile of the formolase and describes alternative routes of carbon assimilation from the pathway. Once this pathway is further evolved, it could supply reduced carbon to engineered polyketide synthase proteins.
Chapter 3 explores the role of the histidine in the GHSxS active site motif of acyl transferase domains. The role of this residue in the literature was unclear, with some reports suggesting that this histidine could even serve as an alternative nucleophile. This study showed that removal of this histidine increases hydrolysis, suggesting it is important for the stabilization of acyl-enzyme intermediates.
Chapter 4 probes the mechanism for gem-dimethylation in complex polyketide biosynthesis. Specifically, this report shows that methylation can precede condensation in polyketide biosynthesis, contrary to the canonical understanding.
Chapter 5 describes the development of the yersiniabactin PKS as a gem-dimethylation catalyst. Specifically, the engineering of this enzyme towards production of several useful gem-dimethylated compounds, as well as the parallel engineering of a thioesterase domain to release gem-dimethylated intermediates is described.
The final chapter will summarize this work, as well as suggesting future efforts to further enable the engineering of polyketide assembly lines.