Elucidation and Engineering of Complete Firefly Luciferin Biosynthesis

萤火虫发光的能力被称为生物发光，这是它们在黑暗中独特的求偶交流的重要特征。自几十年前首次发现以来，萤火虫荧光素-荧光素酶对负责发光反应已广泛应用于农业和生物医学研究。然而，荧光素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。本项目将研究萤火虫荧光素的生物合成。通过本研究确定的新工具将促进自主生物发光的基因工程，而不需要提供外源荧光素。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的时间里对当地约300名K-12学生和约100名高中教师产生积极影响。该项目涉及跨学科的方法，包括生物化学，遗传学，结构生物学和合成生物学来阐明和设计萤火虫的萤光素代谢。为了确定荧光素新生生物合成的代谢途径和酶，将在活体萤火虫中使用一系列同位素标记的示踪剂来鉴定关键的荧光素生物合成中间体。候选酶将通过候选基因方法进行鉴定，并在体外和体内对其生化功能进行表征。该项目进一步寻求确定其他辅助酶，以支持从荧光素酶产物氧荧光素中回收荧光素。有了这些新发现的萤火虫荧光素代谢基因，将在几种模式生物中重建萤火虫的自发发光系统。该项目最终将产生一套新的光遗传学工具，在基础研究和转化研究中都有广泛的应用。该奖项反映了美国国家科学基金会的法定使命，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.