Biosensors for metabolic engineering

用于代谢工程的生物传感器

基本信息

批准号：
7012207
负责人：
JAY D KEASLING
金额：
$ 16.46万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
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/7012207
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

项目摘要

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（由申请人提供）：代谢研究在阐明维持细胞生命的代谢途径方面取得了巨大进步。这项研究产生了代谢物和酶的复杂图谱，描述了生物体中可能发生的化学转化。通过对整个基因组进行测序，可以预见这一阶段研究的结论，从而描述基因组中编码的每个基因产物的功能，并对每种代谢物进行编目。到那时，我们将对一个精致的动态有机体中可能发生的化学现象有一个静态的看法。为了深入了解新陈代谢的动态结构，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节器的天然和工程生物传感器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一般代谢研究具有价值，但我们将把研究重点放在生物传感器的开发上，以指导代谢的进化。传统上，化合物的鉴定依赖于昂贵且笨重的分析仪器，这些仪器需要大量的专业知识才能操作。这些机器生成的数据必须由训练有素的科学家进行解释，才能准确确定复杂样品中是否存在特定化合物。微生物面临着类似的问题，因为它们必须不断地对化学环境进行采样，以确定碳、氮和其他细胞构建材料的最有效来源。微生物通过多种生物传感系统（生物传感器）来完成这项任务，这些系统确定在任何给定时间应该表达哪种代谢途径。利用这些传感器-执行器系统来检测目标化合物不仅将为下一阶段的代谢研究提供工具，而且还将提供指导细胞代谢机制制造目标化合物的方法。这些系统还可用于检测环境中的有毒化学物质，例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实用性，并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用于指导代谢途径的进化，我们将首先使用天然转录调节因子来报告细胞内目标化合物的相对浓度，并且我们将使用该调节因子来指导生物合成途径的进化制备更多的目标化合物。接下来，我们将开发一个选择盒，用于进化具有所需特性的新转录调节因子。最后，我们将使用选择盒来进化新的转录调节因子来检测特定的靶分子。因此，该提案的具体任务是（1）使用内源转录激活剂PrpR来指导丙酸生产途径的进化，（2）构建用于新生物传感器进化的报告/选择（RS）盒，以及(3) 开发新的基于 DitR 的生物传感器来感知不同的目标化合物。