Biosensors for metabolic engineering

用于代谢工程的生物传感器

• 批准号: 6767500
• 负责人: JAY D KEASLING
• 金额: $ 15.91万
• 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/6767500
• 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（由申请人提供）：代谢的研究取得了长足的进步，阐明了维持细胞中生命的代谢途径。 这项研究产生了一个复杂的代谢物和酶图，这些图描述了生物体中可能的化学转化。通过对整个基因组进行测序，可以预见到这一研究阶段的结论，以便将描述每个基因组中每个基因产物的功能，并且将对每个代谢产物进行分类。那时，我们将对精美的动态生物中的化学作用有静态的看法。为了深入了解代谢的动态结构，必须开发新的工具，以测量活细胞中代谢物的瞬时水平。此处提出的研究将开发用于使用基于转录调节器建立的天然和工程生物传感器在体内实时监测体内代谢产物的工具。尽管此处开发的工具对于一般的新陈代谢研究将具有价值，但我们将把研究重点放在生物传感器的开发上，以指导代谢的发展。 传统上，化合物的识别依赖于需要大量专业知识才能运行的昂贵且繁琐的分析工具。这些机器生成的数据必须由训练有素的科学家来解释，以准确确定复杂样品中特定化合物的存在或不存在。微生物面临类似的问题，因为它们必须不断地品尝其化学环境，以确定最有效的碳，氮和其他蜂窝状建筑材料的来源。微生物通过多种生物传感系统（生物传感器）来完成此任务，这些系统（生物传感器）确定应在任何给定时间表达哪种代谢途径。利用这些传感器传导器系统检测目标化合物不仅可以为下一阶段的代谢研究提供工具，而且还将提供方法，以指导细胞的代谢机械以制造靶化合物。这些系统也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的工具的实用性，并开发适用于建造任何所需生物传感器的方法。为了证明可以使用生物传感器来指导代谢途径的演变，我们将首先使用天然转录调节剂来报告细胞内靶化合物的相对浓度，我们将使用该调节剂指导生物合成途径的进化来使靶标的更大。 接下来，我们将开发一个选择盒，以用于具有所需属性的新转录调节器的演变。最后，我们将使用选择盒子进化新的转录调节剂来检测特定的靶标分子。因此，该提案的特定任务是（1）使用内源性转录激活剂PRPR指导丙酸生产途径的演变，（2）以构建一个记者/选择（RS）盒，以发展新的生物传感器，以及（3）进化基于新的DITR的生物传感器，从而觉得不同的靶标的靶标。