NO as a Sensor of Gene Expression Differences: Application to Synthetic Biology

描述（由申请人提供）：合成生物学和代谢工程领域的主要目标之一是通过插入合理工程化的合成基因网络或改变天然存在的代谢网络来重新编程生物体的行为。该过程的一个突出组成部分是需要平衡基因表达以获得所需产物的最佳产量并避免有毒中间体的积累。该提案将通过开发用于分析动态基因表达差异的技术以及用于在代谢途径的背景下解释和校正这些差异的方法来解决目前缺乏满足这一需求的方法的问题。瞬时信号分子一氧化氮（NO）将用于此目的，因为它可以由NO双加氧酶产生并由NO双加氧酶或还原酶降解，从而产生可调信号，该信号将用作两个启动子之间基因表达差异的指示剂。再加上作为本研究的一部分将开发的NO敏感启动子，NO的存在将用于驱动其他酶的基因表达，以响应NO产生和降解水平的不平衡。 作为这个系统的测试，我将把它应用于生产1-丁醇（一种潜在的生物燃料）从苏氨酸通过酮酸生物合成代谢途径。除了将该研究应用于合成生物学和代谢工程之外，开发用于体内的NO传感器将应用于细菌抗生素抗性和哺乳动物细胞信号传导的研究，并且开发平衡基因表达的系统可能应用于基因治疗的研究。 公共卫生相关性：代谢途径和合成网络中基因表达的动态平衡对于所需产品（如生物燃料和生物生产的药物）的调节生产非常重要。这项拟议的研究将提供一种新的工具，用于分析基因表达的差异，并通过使用小信号分子一氧化氮作为表达率的敏感和瞬时指标来平衡各种宿主生物体中的表达水平。该项目的成功完成将提供一个多功能的一氧化氮传感器，可用于调节基因表达，在体内响应一氧化氮，和动态基因表达的分析系统，用于识别和平衡代谢途径中的表达差异。

DESCRIPTION (provided by applicant): One of the primary goals of the fields of synthetic biology and metabolic engineering is to reprogram the behavior of organisms via the insertion of rationally engineered synthetic gene networks or the alteration of naturally occurring metabolic networks. A prominent component of this process is the need to balance gene expression to obtain optimal yields of desired products and avoid buildup of toxic intermediates. This proposal will address the current lack of methodology to serve this need by developing techniques for analyzing differences in dynamic gene expression and methods for accounting for and correcting those differences in the context of a metabolic pathway. The transient signaling molecule nitric oxide (NO) will be used for this purpose because it can be produced by NO synthases and degraded by NO dioxygenases or reductases, resulting in a tunable signal that will serve as an indicator of the difference in gene expression between two promoters. Coupled with a NO-sensitive promoter that will be developed as part of this research, the presence of NO will be used to drive gene expression of additional enzymes in response to an imbalance in the levels of NO production and degradation. As a test of this proposed system, I will apply it towards the production of 1-butanol (a potential biofuel) from threonine via the keto-acid biosynthesis metabolic pathway. In addition to the applications of this proposed research to synthetic biology and metabolic engineering, the development of a NO sensor for use in vivo will have applications to studies of bacterial antibiotic resistance and mammalian cell signaling and the development of a system to balance gene expression may have applications in the study of gene therapy. PUBLIC HEALTH RELEVANCE: Dynamic balancing of gene expression in metabolic pathways and synthetic networks is important for the regulated production of desired products, such as biofuels and biologically produced pharmaceuticals. This proposed research will provide a novel tool for analyzing differences in gene expression and balancing expression levels in a variety of host organisms by use of the small signaling molecule nitric oxide as a sensitive and transient indicator of expression ratios. Successful completion of this project will provide a versatile nitric oxide sensor that can be used to regulate gene expression in response to nitric oxide in vivo, and an analysis system of dynamic gene expression for use in identifying and balancing expression differences in metabolic pathways.