PROJECT SUMMARY The isoprene unit is a structural motif found in >80,000 natural products and is usually critical for biological activity and modulation of pharmacological properties. Yet, our ability to access isoprenoids and diversify their structures has been extremely limited. The complexity of isoprenoid biosynthetic pathways, the difficulty of rationally overcoming multiple critical bottlenecks, and the narrow substrate scope of many components have hampered attempts at forward engineering isoprenoid biosynthesis to expand chemical space. Consequently, the full synthetic potential of isoprenoid biosynthesis has yet to be realized. As part of the long-term goal of reprogramming the biosynthesis of natural products for the synthesis of potential therapeutics, the overall objective of this proposal is to leverage the simplicity, modularity, and versatility of our recently described Alcohol Dependent Hemiterpene (ADH) pathway to isoprenoid building blocks by coupling it to downstream biosynthetic chemistry and applying directed evolution to isoprenoid biosynthesis. Our hypotheses are (1) the simplicity of the ADH pathway facilitates access to isoprenoids, (2) bottlenecks to decorated terpenes can be overcome by directed evolution, and (3) the limited specificity of prenyltransferases can be expanded by directed evolution. These hypotheses are supported by (1) data that validates the ability of the ADH pathway to support production of isoprenoids,26 (2) development of genetically-encoded biosensors for in situ terpene detection, (3) preliminary data that demonstrates the feasibility of enzymatically generating non-isoprene building blocks in vitro and in vivo and coupling them to downstream isoprenoid biosynthesis,27 (4) preliminary data that reveals prenyltransferase promiscuity as a platform for directed evolution, and (5) development of a prenyltransferase high-throughput screen. The rationale for the proposed research is that our approach of directed evolution enables access to a variety of medicinally relevant isoprenoids including new-to-nature compounds. To address these hypotheses, and to complete the overall objective of this proposal, the following specific aims will be completed: (1) overcome bottlenecks to oxygenated terpenes via biosensor-guided engineering and (2) expand isoprenoid chemical diversity via prenyltransferase directed evolution. Our approach is highly innovative because directed evolution of isoprenoid biosynthesis has previously been limited to colorimetric terpenes and our chemo- enzymatic strategy to isoprenoids offers unprecedented scope, versatility, modularity, and utility. The proposed research is significant because it is expected to have broad positive impact in natural product biosynthesis and synthetic biology by advancing new strategies for natural product biosynthesis and enabling access to biologically active natural products not readily accessible by genetic manipulation or conventional organic synthesis...