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

0644678 Cirino这项研究项目通过展示一种通用的方法来开发基于遗传选择或荧光激活细胞分类(FACS)的筛选，以促进从不同的组合文库中快速分离改进的酶或代谢途径变体，从而满足对定制设计的酶和微生物工厂的改进技术的需求。提出了两步法，其中首先使用双重选择系统来设计调节蛋白，从而使感兴趣的产物/代谢物作为转录激活的诱导剂。在第二步中，结合了修改后的调节子的双重选择系统作为高度特异性的报告，将产物形成与可选择的细胞表型相关联。这一方法将以AraC调节子及其同源启动子(“PBAD”)作为模型系统进行演示。这项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，产生新的或改进的生物产品合成。这一计划的更广泛影响在于开发了一种总体上能够推动生物催化进步的技术。重要的是，证明的方法很容易推论到除大肠杆菌以外的微生物。综合教育计划将加强生化工程课程，并为学生提供宝贵的研究经验，为他们在生物技术领域的职业生涯做好准备。计划中的外展工作将通过向任职人数不足以及在教育或经济上处于不利地位的群体提供研究机会，向更广泛的社区促进对科学和工程的热情。

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.