Elucidating the genetic basis of imidazopyrazinone luciferin biosynthesis for the development of improved bioluminescence imaging technologies

阐明咪唑并吡嗪酮荧光素生物合成的遗传基础，以开发改进的生物发光成像技术

• 批准号: 9979650
• 负责人: Tristan de Rond
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2021-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979650
• 关键词: Address; Adoption; Amino Acids; Anabolism; Animal Model; Animals; Biochemical Markers; Biological; Bioluminescence; Biomedical Research; Biotechnology; Candidate Disease Gene; Cell Culture Techniques; Cell membrane; Code; Copepoda; Curiosities; Development; Diffuse; Disease model; Enzymes; Escherichia coli; Fermentation; Fluorescence Microscopy; Gene Expression Profiling; Genes; Genetic; Grant; Green Fluorescent Proteins; Human; Human Cell Line; Image; Imaging technology; In Situ; Left; Light; Luciferases; Malignant Neoplasms; Microscopy; Modeling; Noise; Ostracods; Pathway interactions; Peptide YY; Production; Reagent; Reporter; Reporter Genes; Research; Research Personnel; Saccharomyces cerevisiae; Serum; Signal Transduction; Technology; Testing; Tumor Burden; Variant; Yeasts; Zooplankton; base; bioluminescence imaging; coelenterazine; comparative; contrast imaging; cost; experimental study; extracellular; fluorescence imaging; gene discovery; improved; in vivo; luciferin; metabolic engineering; microscopic imaging; novel; small molecule; transcriptomics

Project Summary / Abstract Coelenterazine and vargulin are naturally-occurring imidazopyrazinone luciferins used in bioluminescence imaging, but their scarcity and need to supply them exogenously to cell culture and animal models limit their use. I propose to elucidate the biosynthesis of these compounds in bioluminescent zooplankton. In Specific Aim 1, I discuss the possible biosynthetic origins for these molecules, and my plans to identify the genes coding for the requisite biosynthetic enzymes. In Specific Aim 2, I describe how I would apply these newly-discovered genes to economically produce coelenterazine by fermentation in S. cerevisiae, addressing a critical barrier to progress. In Specific Aim 3, I propose initial experiments to assess whether the ability to effect the luciferin’s biosynthesis in human cell culture model improves bioluminescence imaging contrast. Successful completion of this project could have revolutionary impacts on biological imaging, akin to how development of Green Fluorescent Protein from a biological curiosity into a multipurpose biochemical marker has radically transformed the way we apply microscopy and imaging to biological questions. Besides improving imaging signal-to-noise, localized in situ luciferin production in cell culture could aid research into inter- and intracellular signaling. Moreover, expression of the luciferin biosynthetic machinery in animals would allow for long-term noninvasive in vivo bioluminescence experiments such as gene expression monitoring and assessing tumor burden in cancer disease models.

项目总结/摘要 腔肠素和vargulin是天然存在的咪唑并吡嗪酮类维生素，用于 生物发光成像，但它们的稀缺性和需要将它们外源地提供给细胞培养 动物模型限制了它们的使用。我建议阐明这些化合物的生物合成， 生物发光浮游动物在具体目标1中，我讨论了 这些分子，以及我的计划，以确定必要的生物合成的基因编码， 内切酶在具体目标2中，我描述了如何将这些新发现的基因应用于 在S.酿酒厂，解决一个关键的 进步的障碍。在具体目标3中，我提出了初步实验，以评估是否有能力 在人类细胞培养模型中影响白蛋白的生物合成改善了生物发光 成像对比度该项目的成功完成可能会产生革命性的影响 生物成像，类似于如何从生物样品中开发绿色荧光蛋白， 好奇心变成了一种多用途的生化标记， 显微镜和成像来解决生物学问题。除了改善成像信噪比， 在细胞培养中的局部原位生产可以帮助研究细胞间和细胞内 发信号。此外，在动物中表达这种生物合成机制将允许 用于长期非侵入性体内生物发光实验， 监测和评估癌症疾病模型中的肿瘤负荷。