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

描述(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设计的合成基因网络或改变自然发生的代谢网络来重新编程生物体的行为。这一过程的一个重要组成部分是需要平衡基因表达，以获得所需产品的最佳产量，并避免有毒中间体的积聚。这项提案将通过开发分析动态基因表达差异的技术，以及在代谢途径背景下解释和纠正这些差异的方法，解决目前缺乏满足这一需求的方法学的问题。瞬时信号分子一氧化氮(NO)将用于这一目的，因为它可以由NO合成酶产生，并被NO双加氧酶或还原酶降解，从而产生一个可调节的信号，作为两个启动子之间基因表达差异的指示器。再加上作为这项研究的一部分将开发的NO敏感启动子，NO的存在将被用来驱动额外酶的基因表达，以应对NO产生和降解水平的失衡。作为对这一系统的测试，我将把它应用于苏氨酸通过酮酸生物合成代谢途径生产1-丁醇(一种潜在的生物燃料)。除了这项拟议的研究在合成生物学和代谢工程中的应用外，体内使用的NO传感器的开发还将应用于细菌抗生素耐药性和哺乳动物细胞信号转导的研究，以及平衡基因表达的系统的开发可能在基因治疗研究中应用。 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的规范生产非常重要，例如生物燃料和生物生产的药品。这项拟议的研究将提供一个新的工具来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比率的敏感和瞬时指标来平衡各种宿主生物的表达水平。该项目的成功完成将提供一个多功能的一氧化氮传感器，可以用来调节体内对一氧化氮的基因表达，以及一个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.