Elucidating and engineering eleutherobin biosynthesis

阐明和工程化刺五加酶生物合成

基本信息

批准号：
10572627
负责人：
Paul Scesa
金额：
$ 12.5万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-04 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10572627
关键词：
Acetates Acids Acyl Coenzyme A Address Agreement Alcohols Anabolism Animals Aquaculture Area Bacteria Basic Science Binding Binding Proteins Biochemical Biochemical Pathway Biochemistry Biological Assay Biological Process Cancer cell line Cells Chemicals Chemotherapy-Oncologic Procedure Chromosomes Creativeness Cyclization Cytochrome P450 Dehydration Diterpenes Engineering Enzymes Escherichia coli Evaluation Fermentation Gene Cluster Genes Goals Growth Harvest Incubated Investigation Laboratories Membrane Mentors Metabolism Methods Microsomes Microtubule stabilizing agent NADPH-Ferrihemoprotein Reductase Natural Products Natural Source Operon Organic Synthesis Oxidases Paclitaxel Pathway interactions Pharmacologic Substance Plasmids Production Proteins Reporting Research Resistance Route Saccharomyces cerevisiae Scientist Series Structure System Technology Terpenes Testing Thermodynamics Training Transferase Work Yeasts amidase analog beta Tubulin biological development career development chemical group chemical synthesis drug discovery eleutherobin farnesyltranstransferase functional group improved innovation marine natural product mevalonate mutant novel oxidation protein aggregation reconstitution success synthetic biology tool

项目摘要

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.

项目概要/摘要 Eleutherobin (1) 是一种从八珊瑚中分离出来的二萜类海洋天然产物 (MNP)。微管稳定剂，1 显示对癌细胞系的生长抑制作用，其效力与紫杉醇，但对 β-微管蛋白突变体的交叉耐药性降低，目前 1 的可持续供应。未通过野生收获、水产养殖或全合成获得 1 的合成生物学方法。已被认为是一种可能的替代方案，但天然途径仍然难以捉摸，因此，生物合成。 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。报道了来自 1, E. caribaerum 生产商的关键萜烯环化酶 EcTPS1 的表征。此外，在动物身上发现 EcTPS1 基因的两侧是预测的氧化酶和酰基转移酶基因这个前所未有的、假定的生物合成基因簇（BGC）为研究提供了明确的方向。重建 1 的生物合成。我们的基本假设是，通过使用我们表征的 EcTPS1 作为我们可以结合化学和酶学方法来生产 1。该提案的总体目标是将前体的异源生产设计为 1，表征在 BGC 中定制酶并将其用于 1 的半合成。这项工作将为通过进一步开发次级代谢工具以及提供商品来促进生物化学领域的发展可持续天然产品供应的形式和新型生物催化剂面临的三个基本挑战。是：1) 不存在针对单胞菌前体的合成生物学途径或其他可持续方法；2) 通过化学合成方法安装含氧官能团需要立体、区域和 3）生物合成途径的定制酶在生化上具有挑战性。这些挑战将通过有机合成和合成生物学来解决。如以下具体目标所述：目标 1) 设计刺五加酶的半合成路线； Subaim 1a) eunicellane 前体 klysimplexin R 的合成生物学途径；Subaim 1b) 化学刺五加酶核心的合成：目标 2) 刺五加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；酰基转移酶的表征这项工作将在 Eric 博士的实验室进行。施密特是一位著名的天然产物生物化学家，将提供优秀的培训和职业生涯让我有机会成为一名成功的、独立的学术科学家，专注于除了我的主要导师施密特博士之外，还有一个由三位杰出人士组成的委员会。科学家 Bradley Moore 博士、Vinayak Agarwal 博士和 Jeffrey Rudolf 博士已同意指导我并将为我实现这些目标的努力提供评估和支持的手段。