Elucidation and Engineering of Complete Firefly Luciferin Biosynthesis

萤火虫产生光的能力，称为生物发光，是一个显着的特征，对于它们在黑暗中进行独特的求偶交流至关重要。自几十年前首次发现以来，负责发光反应的萤火虫荧光素酶对已广泛用于农业和生物医学研究。然而，萤火虫生物发光蛋白生物合成的代谢途径仍然未知，这极大地限制了萤火虫生物发光在更广泛背景下的潜在应用。本计画将探讨萤火虫体内的生物合成。通过这项研究确定的新工具将有助于自主生物发光的基因工程，而无需提供外源性的荧光素。通过生物发光的主题，与此项目相关的科学推广活动也将在三年内对当地约300名K-12学生和约100名高中教师产生积极影响。该项目涉及生物化学，遗传学，结构生物学和合成生物学等跨学科方法，以阐明和设计萤火虫荧光素代谢。为了解析萤火虫素从头生物合成的代谢途径和酶，将在活萤火虫中使用一系列同位素标记的示踪剂来鉴定生物合成中间体中的关键酶。候选酶将通过候选基因的方法进行鉴定，其生化功能将在体外和体内进行表征。该项目进一步寻求鉴定支持从荧光素酶产物荧光素中回收荧光素的其他辅助酶。有了这些新发现的萤火虫荧光素代谢基因，萤火虫自发光系统将在几种模式生物中重建。该项目最终将产生一套新的光遗传学工具，在基础和转化研究中具有广泛的应用。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The ability of fireflies to produce light, called bioluminescence, is a remarkable trait essential for their unique courtship communication in the dark. Since its initial discovery decades ago, the firefly luciferin-luciferase pair responsible for the light-giving reaction has become widely used in agricultural and biomedical research. However, the metabolic pathway underlying luciferin biosynthesis remains unknown, which greatly limits the potential applications of firefly bioluminescence in a broader context. This project will investigate luciferin biosynthesis in fireflies. The new tools identified through this research will facilitate genetic engineering of autonomous bioluminescence without the need of supplying exogenous luciferin. Through the topic of bioluminescence, the scientific outreach activities associated with this project will also positively impact about 300 local K-12 students and about 100 high school teachers over the period of three years.The project involves interdisciplinary approaches including biochemistry, genetics, structural biology, and synthetic biology to elucidate and engineer firefly luciferin metabolism. To resolve the metabolic pathway and enzymes underlying luciferin de novo biosynthesis, a series of isotopically labeled tracers will be used in live fireflies to identify the key luciferin biosynthetic intermediates. The candidate enzymes will be identified through a candidate gene approach, and their biochemical functions will be characterized both in vitro and in vivo. The project further seeks to identify additional accessory enzymes that support luciferin recycling from the luciferase product oxyluciferin. With these newly identified firefly luciferin metabolic genes in hand, the firefly autoluminescent system will be reconstituted in several model organisms. This project will ultimately yield a new set of optogenetic tools with broad applications in both basic and translational research.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.