Elucidating and engineering eleutherobin biosynthesis

项目总结/摘要 五加素（Eleutherobin，1）是从珊瑚中分离得到的一种二萜类海洋天然产物。作为强效 微管稳定剂，1显示出对癌细胞系的生长抑制，其效力与 紫杉醇，但对β-微管蛋白突变体的交叉抗性降低。目前，可持续供应量为1 未通过野生收获、水产养殖或全合成获得。一种合成生物学方法 被认为是一种可能的替代方法，但天然途径仍然难以捉摸。因此， 1提供了一个具有挑战性的研究机会，需要新颖和创造性的想法。最近，我们组 报道了一个关键的萜烯环化酶，EcTPS 1，从生产商的1，E。加勒比海 此外，在动物上发现EcTPS 1基因的侧翼是预测的氧化酶和酰化酶基因 染色体这一前所未有的、推定的生物合成基因簇（BGC）为基因组的进化提供了明确的方向。 重组1的生物合成。我们的基本假设是，通过使用我们表征的EcTPS 1作为 我们可以使用化学和酶方法的组合来生产1。的 该提案的总体目标是设计前体的异源生产，以1，表征 在BGC中定制酶，并将这些酶用于1的半合成。这项工作将提供创新， 通过进一步开发次级代谢的工具以及提供商品， 可持续的天然产品供应和新型生物催化剂的形式。三个基本挑战 目前的努力是：1）不存在合成生物学途径或其他可持续的方法来获得优甲藻烷前体; 2） 通过化学合成手段安装含氧官能团将需要立体、区域和立体化学方法。 化学选择性方法3)生物合成途径的剪裁酶在生物化学上具有挑战性 膜结合蛋白这些挑战将利用有机合成和合成生物学来解决 如以下具体目的所概述的：目的1）工程化一条通向五加苷的半合成路线; Subaim 1a）至eunicellane前体klysimplexin R的合成生物学途径; Subaim 1b）化学 五加苷酶核心的合成：目的2）五加苷酶中剪裁酶的表征 生物合成途径; Subaim 2a）细胞色素P450酶的表征; Subaim 2b） 酰基转移酶的表征。这项工作将在埃里克博士的实验室进行。 施密特，著名的天然产物生物化学家，并将提供一个良好的培训和职业生涯 发展机会，使我成为一个成功的，独立的学术科学家，专注于 生物医学相关领域。除了我的主要导师施密特博士外， 科学家们，布拉德利摩尔博士，维纳亚克阿加瓦尔和杰弗里鲁道夫，已经同意指导我，并将 为我实现这些目标的努力提供一种评估和支持的手段。

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 and support in my effort toward these aims.