Biosensors for metabolic engineering

用于代谢工程的生物传感器

• 批准号: 6844351
• 负责人: JAY D KEASLING
• 金额: $ 16.37万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844351
• 关键词: bioengineering /biomedical engineering; biosensor device; biotechnology; directed evolution; diterpenes; gene induction /repression; genetic regulatory element; green fluorescent proteins; metabolomics; method development; peptide library; propionates; proteomics; site directed mutagenesis; transcription factor

DESCRIPTION (provided by applicant): The study of metabolism has made great strides elucidating the metabolic pathways that sustain life in the cell. This investigation has produced a complex map of metabolites and enzymes that describe the chemical transformations that are possible in an organism. With the sequencing of entire genomes it is possible to foresee the conclusion of this phase of study such that the function of each gene product encoded in a genome will be described and every metabolite will be cataloged. At that point, we will have a static view of the chemistry possible in an exquisitely dynamic organism. To gain insight into the dynamic structure of metabolism, new tools must be developed to measure transient levels of metabolites in a living cell. The research proposed here will develop tools for the real-time monitoring of intra-cellular metabolites in vivo using native and engineering biosensors founded on transcriptional regulators. Although the tools developed here will be of value for the study of metabolism in general, we will focus our research on the development of biosensors for the purpose of directing the evolution of metabolism. The identification of chemical compounds has traditionally relied on expensive and cumbersome analytical instruments that require considerable expertise to operate. The data generated by these machines must be interpreted by a trained scientist to accurately determine the presence or absence of a specific compound in a complex sample. Microorganisms face a similar problem as they must constantly sample their chemical environme nt to identify the most efficient source of carbon, nitrogen and other cellular building materials. Microbes accomplish this task through a multitude of biological sensing systems (biosensors) that determine which metabolic pathway should be expressed at any given time. Exploiting these sensor-actuator systems to detect target compounds will not only provide the tools for the next phase of metabolism study, but will also deliver methodology to direct the metabolic machinery of the cell to make target compounds. These systems also may be used to detect toxic chemicals in the environment such as groundwater contamination or chemical warfare agents. We propose the development of chemical sensor/actuator systems to detect target analytes. We will demonstrate the utility of these biosensors as tools for metabolic engineering, as well as develop methodogy applicable for building any desired biosensor. To demonstrate that a biosensor can be used to direct the evolution of a metabolic pathway, we will first use a native transcriptional regulator to report on the relative concentrations of intracellular target compounds and we will use this regulator to direct the evolution of a biosynthetic pathway to make more of the target compound. Next, we will develop a selection cassette for the evolution of new transcriptional regulators with desired properties. Finally, we will use the selection cassette to evolve new transcriptional regulators to detect specific target molecules. Accordingly, the specific tasks of this proposal are (1) to use the endogenous transcriptional activator PrpR to direct the evolution of a propionate production pathway, (2) to build a reporter/selection (RS) cassette for the evolution of new biosensors, and (3) to evolve new DitR-based biosensors that sense different target compounds.

描述(申请人提供)：新陈代谢研究已经取得了很大进展，阐明了维持细胞内生命的代谢途径。这项研究绘制了一张复杂的代谢物和酶图谱，描述了生物体中可能发生的化学变化。随着整个基因组的测序，有可能预见到这一阶段研究的结论，这样将描述基因组中编码的每一种基因产物的功能，并对每一种代谢物进行分类。在这一点上，我们将有一个静态的观点，在一个精致的动态有机体中可能的化学。为了深入了解代谢的动态结构，必须开发新的工具来测量活细胞中代谢物的瞬时水平。这里提出的研究将开发使用基于转录调控的本地和工程生物传感器来实时监测体内细胞内代谢物的工具。虽然这里开发的工具对于新陈代谢的研究总体上是有价值的，但我们将把我们的研究重点放在生物传感器的开发上，以指导新陈代谢的进化。 化合物的鉴定传统上依赖于昂贵而笨重的分析仪器，这些仪器需要相当多的专业知识才能操作。这些机器产生的数据必须由训练有素的科学家解释，以准确确定复杂样品中特定化合物的存在或不存在。微生物面临着类似的问题，因为它们必须不断地对其化学环境进行采样，以确定碳、氮和其他蜂窝建筑材料的最有效来源。微生物通过许多生物传感系统(生物传感器)完成这一任务，这些系统确定在任何给定的时间应该表达哪种代谢途径。利用这些传感器-致动器系统来检测目标化合物不仅将为下一阶段的新陈代谢研究提供工具，而且还将提供方法学来指导细胞的新陈代谢机制制造目标化合物。这些系统还可用于检测环境中的有毒化学物质，如地下水污染或化学战剂。 我们建议开发化学传感器/执行器系统来检测目标分析物。我们将展示这些生物传感器作为代谢工程工具的效用，以及开发适用于构建任何所需生物传感器的方法。为了证明生物传感器可以用来指导代谢途径的进化，我们首先将使用一个天然的转录调节因子来报告细胞内目标化合物的相对浓度，我们将使用这个调节因子来指导生物合成途径的进化，以使目标化合物更多。下一步，我们将开发一种选择盒式磁带，用于进化具有所需特性的新转录调控因子。最后，我们将使用选择盒进化出新的转录调节因子来检测特定的靶分子。因此，这个建议的具体任务是(1)使用内源转录激活因子PRPR来指导丙酸产生途径的进化，(2)构建一个报告/选择(RS)盒来进化新的生物传感器，以及(3)进化新的基于DitR的生物传感器来感知不同的目标化合物。