SUMMARY ABSTRACT Natural products are potently active, privileged scaffolds that form the basis of our therapeutic arsenal across all areas of human health. The continued development of natural products and their analogs will provide access to compounds with improved activity and pharmacological properties while decreasing off-target effects. The bioengineering of individual biosynthetic enzymes is one method of generating such novel secondary metabolites. However, bioengineering efforts are often stymied due to a lack of fundamental understanding of the discrete enzymatic transformations responsible for natural product biosynthesis. Likewise, whole pathway metabolic engineering focused on generating novel secondary metabolites with targeted structural alterations requires detailed knowledge of individual biosynthetic steps. Fatty acyl-AMP ligases (FAALs) are pivotal biosynthetic domains that draw fatty acids from primary metabolism for incorporation into more complex natural product scaffolds. The FAAL domains are often linked with multidomain polyketide synthases either in cis or trans via structural linker regions or docking domains, respectively. We hypothesize that these linker regions and docking domains are crucial to the transfer of fatty acid chains of specific lengths to the downstream polyketide synthase domains and that we can modulate this transfer by maintaining the appropriate key elements. This proposal seeks to identify the key residues that control the activation and transfer of fatty acid chains in a model system for application to more complex pathways. As well, we seek to develop a robust heterologous host capable of producing these fatty acid-containing metabolites. In Aim I, we will use our model system olefin (Ols) synthase to identify and modulate the gate-keeping linker regions and docking domains that govern fatty acid integration into secondary metabolites. Directed evolution experiments using the bacterial two-hybrid system will allow us to dissect the key docking domains found in Ols homologs that contain a trans enzymatic structure. In a complementary system, we will perform directed evolution experiments targeted towards the linker regions of cis Ols synthases and directly assess metabolite production via a temperature selection screening. Aim I will uncover the key structural elements in the model Ols synthase for future bioengineering of more complex natural product enzymes with similar biosynthetic logic. In Aim II, we propose to develop Pseudomonas putida for the heterologous expression of fatty acid- containing natural products. The pathways for our model Ols synthase as well as the biosynthetic gene cluster encoding for micacocidin production will be expressed in P. putida. The production and bioengineering of the FAAL-ACP domains to integrate acyl chains of varying length will be encoded in this heterologous host to facilitate engineering efforts. The bioengineering of the FAAL-ACP loading modules in Ols...