PROJECT SUMMARY The complexity of polyketide biosynthetic machinery has hampered attempts to access macrolides and their analogs via combinatorial biosynthesis. As part of our long-term goal of reprogramming the biosynthesis of natural products for the synthesis of therapeutic leads, the overall objective here is to use genetically encoded biosensors to enhance access to novel macrolides. Our hypotheses are (1) the established inducer promiscuity of the MphR repressor protein can be manipulated to provide biosensors with new inducer specificities and selectivities, (2) the specificity of macrolide tailoring enzymes can be manipulated by biosensor-guided directed evolution, and (3) MphR can be used to identify hybrid assembly lines with improved activities. These hypotheses are supported by (1) preliminary data that shows MphR variants with new specificities, selectivities, and suitable detection capabilities can be generated, (2) the variety of macrolide tailoring enzymes available as starting points for directed evolution and the success of directed evolution for altering substrate specificity of other enzymes, and (3) preliminary data that demonstrates the feasibility of using trans-acting enzymes to complement polyketide assembly lines. The rationale for the proposed research is that our approach of leveraging designer biosensors offers the ability to report the activity of a variety of macrolide biosynthetic enzymes, which can be applied to solving a broad range of problems related to macrolide biosynthesis, leading to valuable new macrolides. To address these hypotheses, and to complete the overall objective of this proposal, the following specific aims will be completed: (1) access novel macrolide O-alkyl derivatives, and (2) rescue the activity of poorly active hybrid PKS assembly lines. Our approach is highly innovative because it develops a set of screening tools that are currently not available and that can be applied to engineering the biosynthesis of a broad range of macrolides in potentially any microbial host. The proposed research is significant because it is expected to have broad positive impact in natural product biosynthesis and synthetic biology by developing new strategies for producing macrolides, by expanding our understanding of biosensor specificity, by developing new approaches for macrolide diversification, and by expanding the capabilities of enzyme engineering and synthetic biology.