Collaborative Research: RUI: Broadening the Application of Programmed Evolution for Metabolic Engineering

代谢工程中的设计考虑是基于对作用于工程细菌种群的进化力的不完全理解。这个项目通过利用进化而不是对抗进化来颠覆代谢工程范式。研究人员开发了一种代谢工程的进化方法，使细菌能够整合它们的生长环境和它们的工程代谢。这种方法被称为程序化进化，因为细菌种群被DNA软件编程，以计算代谢途径优化问题的解决方案，进化被用来指导细菌种群走向最佳解决方案。该项目的目标是通过开发一种发现新核糖开关的新方法来扩展程序化进化，使用新的核糖开关优化表达生产新化合物所需的酶，并开发数学模型和计算工具来支持这两个过程。这些方法将降低在代谢工程细菌中生产用于能源、制药和生物修复的有用化合物的成本。该项目将增加科学教育的多样性，有助于培养具有竞争力和科学素养的劳动力，并有可能提高美国的经济竞争力。该项目响应了《愿景与变革》（Vision and Change）的号召，要求本科生提出研究问题，提出可测试的假设，收集和分析数据，并交流结果，从而获得更真实的本科生研究经验。当他们学习如何为代谢工程编程细菌的进化时，本科研究生将学习如何为他们自己的未来编程，成为具有科学素养的公民、教育工作者和研究科学家。程序化进化是一种优化细菌正交代谢途径的模块化系统。它使用组合学、适应性和生物传感器模块，这些模块可以单独开发和测试，组合使用，并在研究小组之间共享。程序化进化的一个关键组成部分是核糖体开关，它将代谢输出转化为适应性基因表达和选择优势。大多数用于代谢工程的核糖开关都包含通过体外指数富集配体系统进化过程发现的RNA适体。然而，体外发现的适体很少在体内起作用。研究人员建议开发基于细胞的指数富集作为一种新的体内方法来发现细菌细胞中可预测功能的核糖开关。该方法通过引入核糖开关的遗传变异来建立文库，应用阴性和阳性选择，并对新核糖开关的表型和基因型进行表征。这种新方法的重要意义在于，它有可能推动人们对天然存在的核糖开关的认识，并发现新的核糖开关，用于能源、制药和生物修复。

Design considerations during metabolic engineering have been based on incomplete understanding of the evolutionary forces acting upon populations of engineered bacteria. This project inverts the metabolic engineering paradigm by harnessing evolution instead of fighting it. The investigators developed an evolutionary approach to metabolic engineering that enables bacteria to integrate their growth environment and their engineered metabolism. The approach is called Programmed Evolution because a population of bacteria is programmed with DNA software to compute solutions to a metabolic pathway optimization problem, and evolution is used to direct the bacterial population toward optimal solutions. The goal of this project is to expand Programmed Evolution by developing a new method for new riboswitch discovery, using the new riboswitches optimally express the enzymes necessary for e the production of new compounds, and developing mathematical models and computational tools to support both processes. These approaches will reduce the cost of producing useful compounds in metabolically engineered bacteria for applications in energy, pharmaceuticals, and bioremediation. This project will increase diversity for science education, contribute to a competitive and scientifically literate workforce, and has the potential to improve American economic competitiveness. This project responds to the call in Vision and Change for more authentic undergraduate research experiences as undergraduates pose research questions, develop testable hypotheses, collect and analyze data, and communicate results. As they learn how to program the evolution of bacteria for metabolic engineering, undergraduate research students will learn how to program the course of their own futures as science literate citizens, educators, and research scientists.Programmed Evolution is a modular system for the optimization of orthogonal metabolic pathways in bacteria. It uses combinatorics, fitness, and biosensor modules that can be developed and tested separately, used in combinations, and shared among research groups. A key component of Programmed Evolution is the riboswitch that transduces metabolic output into fitness gene expression and selective advantage. Most riboswitches used in metabolic engineering incorporate RNA aptamers discovered by the in vitro process of Systematic Evolution of Ligands by Exponential enrichment. However, aptamers discovered in vitro rarely function in vivo. The investigators propose to develop Cell-based Exponential enrichment as a new in vivo method of discovering riboswitches that function predictably in bacterial cells. The new method introduces genetic variation in a riboswitch to produce a library, applies negative and positive selection, and characterizes the phenotype and genotype of new riboswitches. The significance of the new approach derives from its potential to advance knowledge of naturally occurring riboswitches and to discover new riboswitches for applications in energy, pharmaceuticals and bioremediation.