Project Summary/Abstract Eleutherobin (1) is a diterpenoid marine natural product (MNP) isolated from octocorals. As a potent microtubule stabilizing agent, 1 shows growth inhibition toward cancer cell lines with potency comparable to paclitaxel but with reduced cross-resistance toward β-tubulin mutants. Currently, a sustainable supply of 1 has not been accessed through wild harvest, aquaculture or total synthesis. A synthetic biology approach toward 1 has been considered as a possible alternative, but the native pathway remains elusive. Thus, the biosynthesis of 1 provides a challenging research opportunity in need of novel and creative ideas. Recently, our group has reported the characterization of a key terpene cyclase, EcTPS1, from a producer of 1, E. caribaeorum. Furthermore, the EcTPS1 gene was found to be flanked by predicted oxidase and acylase genes on an animal chromosome. This unprecedented, putative biosynthetic gene cluster (BGC) provides a clear direction for reconstituting biosynthesis of 1. Our underlying hypothesis is that by using our characterized EcTPS1 as a starting point we can produce 1 using a combination of chemical and enzymological methods. The overall goal of this proposal will be to engineer heterologous production of precursors to 1, characterize the tailoring enzymes in the BGC and employ these in a semi-synthesis of 1. This work will provide innovation in the field biochemistry by further developing tools in secondary metabolism as well as affording commodities in the form of sustainable natural product supply and novel biocatalysts. Three essential challenges toward these efforts are: 1) No synthetic biology route or other sustainable approach to a eunicellane precursor exists; 2) Installation of oxygenated functional groups by chemical synthetic means will require stereo-, regio- and chemoselective methods. 3) The tailoring enzymes of the biosynthetic pathway are biochemically challenging membrane bound proteins. These challenges will be addressed using organic synthesis and synthetic biology as outlined in the following specific aims: Aim 1) Engineering a semi-synthetic route toward eleutherobin; Subaim 1a) Synthetic biology route to the eunicellane precursor klysimplexin R; Subaim 1b) Chemical synthesis of the eleutherobin core: Aim 2) Characterization of tailoring enzymes in the eleutherobin biosynthetic pathway; Subaim 2a) Characterization of cytochrome P450 enzymes; Subaim 2b) Characterization of acyl transferase enzymes. This work will be conducted in the laboratory of Dr. Eric Schmidt, a renowned natural products biochemist, and will provide an excellent training and career development opportunity for me to become a successful, independent academic scientist focusing on biomedically relevant areas. In addition to my primary mentor Dr. Schmidt, a committee of three prominent scientists, Drs. Bradley Moore, Vinayak Agarwal and Jeffrey Rudolf, have agreed to mentor me and will provide a means of evaluation an...