CAREER: Engineering Regulatory Protein Effector Specificity to Facilitate Combinatorial Library Analyses

职业：工程调节蛋白效应子特异性以促进组合文库分析

• 批准号: 1135710
• 负责人: Patrick Cirino
• 金额: $ 12.76万
• 依托单位: University of Houston
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1135710&HistoricalAwards=false
• 关键词: CAREER; Engineering; Regulatory; Protein; Effector

0644678CirinoThis research project addresses the need for improved technologies for engineering custom-designed enzymes and microbial factories by demonstrating a general approach to developing genetic selections or fluorescence-activated cell sorting (FACS) based screens to facilitate the rapid isolation of improved enzyme or metabolic pathway variants from diverse combinatorial libraries. Two-step approach was proposed, where a dual selection system is first used to engineer a regulatory protein such that the product/metabolite of interest inimitably acts as inducer of transcriptional activation. In the second step, the dual selection system incorporating the modified regulator serves as a highly specific reporter, correlating product formation with a selectable cellular phenotype. This approach will be demonstrated using the AraC regulator and its cognate promoter ("PBAD") as a model system. The proposed research will demonstrate the integration of protein engineering and directed evolution with metabolic engineering, yielding novel or improved synthesis of bioproducts. The broader impacts of this plan lie in the development of a technology generally enabling advances in biocatalysis. Importantly, the demonstrated method is readily extrapolated to microorganisms other than E. coli. The integrated education plan will strengthen the biochemical engineering curriculum and provide invaluable research experiences to students, preparing them for careers in biotechnology. Planned outreach efforts will promote enthusiasm in science and engineering to a broader community by providing research opportunities to underrepresented and educationally or economically disadvantaged groups.

0644678Cirino该研究项目通过展示开发基于遗传选择或荧光激活细胞分选（FACS）的筛选的通用方法，以促进快速分离改进的酶或代谢途径变体，解决了对工程定制酶和微生物工厂改进技术的需求来自不同的组合库。提出了两步方法，其中首先使用双重选择系统来设计调节蛋白，使得感兴趣的产物/代谢物独特地充当转录激活的诱导剂。在第二步中，结合了修饰调节剂的双重选择系统充当高度特异性的报告基因，将产物形成与可选择的细胞表型相关联。该方法将使用 AraC 调节器及其同源启动子（“PBAD”）作为模型系统进行演示。拟议的研究将展示蛋白质工程和定向进化与代谢工程的整合，产生新颖或改进的生物产品合成。该计划更广泛的影响在于开发一种普遍促进生物催化进步的技术。重要的是，所证明的方法很容易外推到大肠杆菌以外的微生物。综合教育计划将加强生物化学工程课程，并为学生提供宝贵的研究经验，为他们从事生物技术职业做好准备。 计划外展工作将通过为代表性不足和教育或经济弱势群体提供研究机会，提高更广泛社区对科学和工程的热情。