Elucidation and Engineering of Complete Firefly Luciferin Biosynthesis

完整萤火虫荧光素生物合成的阐明和工程

• 批准号: 1818132
• 负责人: Jing-Ke Weng
• 金额: $ 70万
• 依托单位: Whitehead Institute for Biomedical Research
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1818132&HistoricalAwards=false
• 关键词: Elucidation; Engineering; Complete; Firefly; Luciferin

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.

萤火虫产生光的能力（称为生物发光）是它们在黑暗中独特的求偶交流所必需的显着特征。自从几十年前首次发现以来，负责发光反应的萤火虫荧光素-荧光素酶对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。该项目将研究萤火虫中的荧光素生物合成。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，而无需提供外源荧光素。通过生物发光主题，与该项目相关的科学推广活动还将在三年内对约 300 名当地 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。为了解析荧光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识别关键的荧光素生物合成中间体。将通过候选基因方法鉴定候选酶，并在体外和体内表征它们的生化功能。该项目进一步寻求识别其他辅助酶，支持从荧光素酶产品氧化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，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，在基础研究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智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Evidence for de novo Biosynthesis of the Luminous Substrate Coelenterazine in Ctenophores

• DOI: 10.1016/j.isci.2020.101859
• 发表时间: 2020-12-10
• 期刊: iScience
• 影响因子: 5.8
• 作者: Bessho-Uehara M;Huang W;Patry WL;Browne WE;Weng JK;Haddock SH
• 通讯作者: Haddock SH