Elucidating and engineering eleutherobin biosynthesis

阐明和工程化刺五加酶生物合成

• 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
• 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

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.

项目摘要/摘要 元素（1）是一种从八角形分离的数字海洋天然产物（MNP）。作为有效的 微管稳定剂，1显示了对癌细胞系的生长抑制，其效力可与 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1 无法通过野生收获，水产养殖或总合成来进入。一种合成生物学方法1 被认为是可能的替代方法，但本地途径仍然难以捉摸。那是生物合成 1的挑战提供了需要新颖和创意的研究机会。最近，我们的小组有 报道了来自1，Caribaeorum的生产商的键萜烯环酶EctPS1的表征。 此外，在动物上发现ECTPS1基因在预测的氧化酶和酰基酶基因的两侧 染色体。这个前所未有的假定生物合成基因簇（BGC）为 重建1的生物合成。我们的基本假设是，将我们的特征ECTPS1用作 一个起点我们可以使用化学和酶学方法的组合产生1个。这 该提案的总体目标是设计前体的异源生产至1 调整BGC中的酶，并在1的半合成中使用它们。这项工作将提供创新 通过进一步开发二级代谢的工具以及为商品提供商品，该领域生物化学 可持续的天然产品供应和新型生物催化剂的形式。对这些的三个基本挑战 努力是：1）不存在合成生物学途径或其他可持续的前体前体的可持续方法； 2） 通过化学合成手段安装氧化官能团将需要立体声，区域和 化学选择性方法。 3）生物合成途径的裁缝酶在生化上挑战 膜结合蛋白。这些挑战将使用有机合成和合成生物学解决 如以下特定目的所述：目标1）迈向半合成途径迈向元素病因； Subaim 1A）综合生物学途径通往Eunicellane的前体klysimplexin r; Subaim 1b）化学 元蛋白核心核心的合成：目标2）蛋白酶中剪裁酶的表征 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Eric博士的实验室进行。 施密特（Schmidt）是一位著名的天然产品生物化学家，将提供出色的培训和职业 我成为一个成功的独立学术科学家的发展机会，专注于 生物医学相关领域。除了我的主要导师施密特博士，这是一个由三个著名的委员会组成的委员会 科学家，博士。布拉德利·摩尔（Bradley Moore），维纳亚克·阿加瓦尔（Vinayak Agarwal）和杰弗里·鲁道夫（Jeffrey Rudolf）已同意指导我 在我实现这些目标的努力中，提供评估和支持的方法。