CAREER: Development of 13C-assisted Metabolic Flux Analysis Tools for Metabolic Engineering of Cyanobacteria

蓝藻拥有一个复杂而独特的碳代谢网络，使它们能够在水生环境中以最小的营养物质茁壮成长。本研究开发并应用13c辅助代谢通量分析工具，对模式蓝藻Synechocystis sp. PCC6803和Cyanothece 51142的光自养代谢进行了研究。研究目标包括：1)改进基于gc - ms的代谢物和同位素异构体（同位素异构体）测量技术；2)建立基于快速代谢代谢物中同位素分布的动态通量模型，以解析碳通量和代谢调控；3)将通量模型应用于蓝藻代谢工程，通过非发酵途径合成丁醇。本研究将生成13c辅助通量分析工具，以了解蓝藻中酶的功能和代谢调节。这项研究还将开发利用二氧化碳和阳光生产生物燃料的菌株。更广泛的影响本项目探索蓝藻的应用，为能源和环境危机提供潜在的解决方案。本项目开发的13c辅助通量分析工具可用于研究许多其他光合生物，包括用于商业农业的植物。此外，研究和教育工作将通过以下方式结合起来：1)将开设两门生物技术课程（生物过程实验室和合成生物学），以提高本科生的生物实验室技能和生物科学知识；2) CAREER研究将与华盛顿大学iGEM（国际基因工程机器竞赛）团队项目相结合，鼓励女性/少数民族学生参与；3) CAREER研究将与华盛顿大学清洁煤项目（由圣路易斯当地公司支持）相结合，为学生提供工业经验。

Cyanobacteria possess a complex and unique network for carbon metabolism, enabling them to thrive on the minimal nutrients available in aquatic environments. This research develops and applies 13C-assisted metabolic flux analysis tools to study photoautotrophic metabolism in the model cyanobacteria Synechocystis sp. PCC6803 and Cyanothece 51142. The research objectives include: 1) improving GC-MS-based metabolite and isotopomer (isotopic isomer) measurement techniques, 2) developing a dynamic flux model to decipher carbon fluxes and metabolic regulation based on isotopomer distribution in fast-turnover metabolites, 3) applying the flux model for metabolic engineering of cyanobacteria to synthesize butanol via non-fermentative pathways. This research will generate 13C-assisted flux analysis tools to understand enzyme functions and metabolic regulations in cyanobacteria. This study will also develop strains to produce biofuels from carbon dioxide and sunlight.Broader ImpactsThis project explores cyanobacterial applications, offering a potential solution to both energy and environmental crises. The 13C-assisted flux analysis tools developed in this project can be used to investigate many other photosynthetic organisms including plants used for commercial agriculture. Furthermore, research and education efforts will be combined through the following: 1) Two biotechnology courses (Bioprocess Laboratory and Synthetic Biology) will be created to improve both biological laboratory skills and bioscience knowledge for undergraduate students; 2) the CAREER research will be integrated with the Washington University iGEM (International Genetically Engineered Machine Competition) team project, encouraging participation of female/minority students; 3) the CAREER research will be integrated with the Clean-Coal Program at Washington University (supported by local companies in St. Louis) to provide students with industrial experience.