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 Eleutherobin(1)是从八珊瑚中分离得到的一种二萜海洋天然产物(MNP)。作为一种强有力的 微管稳定剂1对癌细胞生长有抑制作用，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的可持续供应具有 不是通过野生收获、水产养殖或完全合成获得的。1的合成生物学方法 已经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 Of 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，我们小组已经 报道了一种产自加勒比乳杆菌(E.caribaorum)的关键萜类环化酶EcTPS1的特性。 此外，在动物身上发现EcTPS1基因两侧有预测的氧化酶和酰基酶基因。 染色体。这一史无前例的、假定的生物合成基因簇(BGC)为 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EcTPS1作为 作为起点，我们可以使用化学方法和酶学方法相结合的方法来生产1。这个 这项提议的总体目标将是将前体的异源生产设计为1，表征 在BGC中剪裁酶并将其用于1的半合成。这项工作将在 通过进一步开发次生代谢工具以及在 可持续的天然产品供应形式和新型生物催化剂。面临的三个基本挑战 努力是：1)不存在合成生物学路线或其他可持续的方法来获得环烷前体；2) 通过化学合成方法安装含氧官能团将需要立体、区域和 化学选择方法。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。这些挑战将通过有机合成和合成生物学来解决。 具体目标如下：目标1)设计一条半合成路线，以获得埃鲁瑟罗宾； 苏巴伊姆1a)合成生物路线，获得尤尼西林前体klyimplexin R；苏巴伊姆1b)化学 Eleutherobin核心的合成：目的2)Eleutherobin中剪裁酶的特性 生物合成途径；Subaim 2a)细胞色素P450酶的特性 酰基转移酶的特性。这项工作将在埃里克博士的实验室进行 施密特，著名的天然产品生物化学家，将提供出色的培训和职业生涯 我有机会成为一名成功的、独立的学术科学家，专注于 与生物医学相关的领域。除了我的主要导师施密特博士，一个由三位杰出人物组成的委员会 科学家布拉德利·摩尔博士、维纳亚克·阿加沃博士和杰弗里·鲁道夫博士已同意指导我和威尔 为我实现这些目标的努力提供一种评估和支持的手段。