Collaborative Research: Simplifying metabolic pathways by wiring redox proteins together

合作研究：通过将氧化还原蛋白连接在一起简化代谢途径

• 批准号: 1402913
• 负责人: Scott Banta
• 金额: $ 30万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402913&HistoricalAwards=false
• 关键词: Collaborative; Research; Simplifying; metabolic; pathways

PI's: Koder, Ronald / Banta, Scott A.Proposal Numbers: 1403748 / 1402913Institutions: CUNY City College / Columbia UniversityTitle: Collaborative Research: Simplifying metabolic pathways by wiring redox proteins together Energy transfer between enzymatic proteins is often accomplished using small molecule cofactors, such as NAD(H) and NADP(H). This system is advantageous in biological systems; however, in technology applications the use of these cofactors is undesirable for several reasons including their high cost and low stability. Advances in protein design and protein engineering have enabled tremendous advances in synthetic biology where new proteins with novel, unnatural functions can be created and characterized. In this project a new protein will be made by coupling two cofactor-dependent enzymes together. In order to eliminate the need for the cofactor, the enzymes will be connected with a designed 'staple' peptide system that contains redox center that enable electron transfer between the enzymes. This will 'wire' the proteins together so that neither one requires the unstable cofactor molecules for activity. Combined, this new enzyme complex will have an activity that is not found in nature. And this will chart a path forward for wiring other redox proteins together towards a goal of eliminating cofactor requirements in future synthetic biology projects. The team consists of PIs from City College of New York and Columbia University. There will be several outreach activities associated with this project including interactions with local high school teachers and students.The overall goal of this collaborative project is to use protein engineering to wire two unrelated redox proteins together creating a novel cofactor-less enzymatic reaction. The investigators will attempt to demonstrate the first example of the wiring of two redox proteins together to form a ping pong redox enzyme with an activity not found in nature. They will start with two known redox enzymes: formate dehydrogenase and lactate dehydrogenase. These enzymes will be connected by a novel 'collagen staple' system that will enable specific self-assembly and the introduction of redox centers for direct electron transfer between the two enzyme active sites. The final enzyme will have a completely novel function: pyruvate reduction to lactate with the concomitant oxidation of formate to carbon dioxide. The new kinetic mechanism and activity of the resultant biocatalyst will be extensively characterized. This approach can then be extended to combine additional enzymes, creating new cofactor-less biocatalysts for industrially important oxidation and reduction reactions. This approach will allow to combine redox enzymes with sequential kinetic mechanisms into a single ping pong enzyme with a synthetic redox center. The research is integrated with educational activities which introduces undergraduates, graduate students and New York City Public High School teachers to the interdisciplinary science of biophysics. These efforts will encourage young students to pursue careers in Science, Technology, Engineering and Mathematics.This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.

PI的：Koder，Ronald / Banta，Scott A. propopal数字：1403748 / 1402913INSTITITS：CUNY CITY COLLECAL / COLUMBIA UNIXICATITLE：协作研究：通过使用小型分子cofactors（例如，H）来简化代谢途径，通过将氧化还原蛋白通过酶蛋白之间的能量转移进行连接在一起，而不是通过酶蛋白之间的成就（H）。 该系统在生物系统中是有利的。但是，在技术应用中，这些辅助因子的使用是不希望的，原因是它们的高成本和低稳定性。 蛋白质设计和蛋白质工程的进步已经在合成生物学方面取得了巨大进步，在这些蛋白质生物学中，可以创建和表征具有新颖，不自然功能的新蛋白质。 在这个项目中，将通过将两个辅助因子依赖性酶耦合在一起制成新蛋白质。 为了消除对辅因子的需求，将与设计的“主食”肽系统连接酶，该肽系统包含氧化还原中心，该氧化还原中心能够在酶之间进行电子转移。 这将将蛋白质“线”“线接线”，以便两者都不需要不稳定的辅因子分子才能进行活性。 结合使用，这种新酶复合物将具有自然界中未发现的活性。 这将列出将其他氧化还原蛋白接线的前进道路，以取消在未来的合成生物学项目中消除辅助因子需求的目标。 该团队由纽约市城市学院和哥伦比亚大学的PI组成。 该项目将有一些与当地高中教师和学生的互动相关的外展活动。该协作项目的总体目的是使用蛋白质工程将两种无关的氧化还原蛋白接种在一起，从而形成一种新型的无辅助因子酶促反应。 研究人员将尝试证明两种氧化还原蛋白接线的第一个例子，以形成乒乓氧化还原酶，具有自然界中未发现的活性。 它们将从两种已知的氧化还原酶开始：甲酸甲酸盐脱氢酶和乳酸脱氢酶。 这些酶将通过一种新型的“胶原蛋白主食”系统连接，该系统将使特定的自组装和引入两个酶活性位点之间直接电子转移的氧化还原中心。 最终酶将具有完全新颖的功能：丙酮酸还原与甲酸甲酯与二氧化碳的氧化作用。 最终的生物催化剂的新动力学机制和活性将得到广泛的特征。 然后可以扩展这种方法以结合其他酶，从而为工业上重要的氧化和还原反应创建新的无辅助因子生物催化剂。这种方法将允许将氧化还原酶与顺序动力学机理结合到单个乒乓酶与合成氧化还原中心。 这项研究与介绍本科生，研究生和纽约市公立高中教师的教育活动融合了生物物理学跨学科科学。 这些努力将鼓励年轻学生从事科学，技术，工程和数学的职业。该奖项由CBET部门的生物技术，生化和生物量工程计划颁发，由分子和细胞生物学系的系统和合成生物学计划共同资助。