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

项目摘要/摘要 长春花碱和精氨酸是天然存在的咪唑并吡嗪酮类荧光素，用于 生物发光成像，但它们的稀缺性需要将其外源供应给细胞培养 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在 会发光的浮游动物。在具体目标1中，我讨论了可能的生物合成来源 这些分子，以及我计划识别编码必要的生物合成的基因 酵素。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 在酿酒酵母中通过发酵经济地生产coelenterazine，解决了一个关键的问题 前进的障碍。在具体目标3中，我提出了初步的实验来评估能力 在人细胞培养模型中影响荧光素的生物合成提高生物发光 成像对比度。这个项目的成功完成可能会对 生物成像，类似于如何从生物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，从根本上改变了我们应用 对生物学问题的显微镜和成像。除了提高成像信噪比外， 在细胞培养中定位原位生产荧光素有助于细胞内和细胞内的研究 发信号。此外，在动物体内表达荧光素生物合成机制将允许 用于长期无创的体内生物发光实验，如基因表达 监测和评估癌症疾病模型中的肿瘤负担。

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.