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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 /摘要鞘嗪和瓦尔古林是自然存在的咪唑吡嗪酮荧光素生物发光成像，但是它们的稀缺性并需要外源性地向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的浮游动物。在特定目标1中，我讨论了可能的生物合成起源这些分子以及我识别编码必要生物合成的基因的计划酶。在特定目标2中，我描述了如何将这些新发现的基因应用于经济上通过在酿酒酵母的发酵来产生的鞘嗪，解决了关键进步的障碍。在特定目标3中，我提出了初步实验来评估能力是否能力在人类细胞培养模型中实现荧光素的生物合成可改善生物发光成像对比。成功完成该项目可能会对生物成像，类似于如何从生物学中发育的绿色荧光蛋白好奇心成多功能生化标记物已从根本上转变我们的应用方式显微镜和成像为生物学问题。除了改善成像信号到噪声外，细胞培养中的原位原位荧光素产生可以帮助研究细胞内和细胞内信号。此外，动物中荧光素生物合成机械的表达将允许长期无创的体内生物发光实验，例如基因表达监测和评估癌症模型中的肿瘤燃烧。