Metabolic Engineering (ME): Genomic and Genetic Approaches to Solvent Tolerance in Anaerobes

代谢工程（ME）：厌氧菌溶剂耐受性的基因组和遗传学方法

• 批准号: 0331402
• 负责人: Eleftherios Papoutsakis
• 金额: $ 60.75万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0331402&HistoricalAwards=false
• 关键词: Metabolic; Engineering; ME; Genomic; Genetic

This project is to understand and exploit the molecular basis that determines tolerance of the industrially important anaerobic clostridia to solvents. Furthermore, the Principal Investigators (PIs) aim to develop general genomic and metabolic engineering strategies for understanding the molecular basis of tolerance to chemicals and for developing tolerant strains. The PIs' hypothesis is that the molecular basis of what makes bacterial cells able to withstand high solvent concentrations can be used to metabolically engineer cells so that they can tolerate higher concentrations of solvents and related chemicals. For these studies, the PIs will employ Clostridium acetobutylicum, whose genome has been sequenced. Based on data from the current funding period, overexpression of heat-shock (stress) proteins (HSPs) - which are molecular chaperones assisting protein folding and re-folding - was shown to confer increased ability to produce and tolerate high butanol concentrations, as well as prolonged cell metabolism. These traits were likely due to the stabilizing effect of HSPs on the cellular machinery. A first specific aim of this project is to use the concomitant overexpression of HSPs and metabolic genes for generating strains, which produce and tolerate high levels of butanol. Promising recombinant strains will be characterized in fermentation studies using DNA-array based transcriptional, Western and flux analysis. A second objective is to use DNA arrays and a "Systems Biology" approach for identifying other genes that might be involved in and confer solvent tolerance. The two proposed strategies constitute Aims 2 and 3. The first strategy involves the construction of plasmid-based genomic libraries (with different origins of replication), transformation into the parent organism, and identification of those plasmids/genes, which confer the desirable phenotype. This approach builds upon and extends a recently proposed method and employs a DNA-array based selection process. Second, full-genome DNA arrays will be used to capture the global picture of the transcriptional programs of four different solvent-tolerant strains in order to identify commonly overexpressed genes possibly related to the tolerant phenotype.

该项目旨在了解和利用决定工业上重要的厌氧梭菌对溶剂的耐受性的分子基础。此外，首席研究员 (PI) 旨在开发通用基因组和代谢工程策略，以了解化学品耐受性的分子基础并开发耐受菌株。 PI 的假设是，细菌细胞能够耐受高溶剂浓度的分子基础可用于对细胞进行代谢工程，使其能够耐受更高浓度的溶剂和相关化学品。对于这些研究，PI 将使用丙酮丁醇梭菌，其基因组已被测序。根据当前资助期间的数据，热休克（应激）蛋白（HSP）的过度表达（HSP是协助蛋白质折叠和重新折叠的分子伴侣）被证明可以增强生产和耐受高浓度丁醇的能力。由于细胞新陈代谢时间延长。这些特征可能是由于热休克蛋白对细胞机制的稳定作用所致。该项目的第一个具体目标是利用热休克蛋白和代谢基因的伴随过度表达来产生菌株，这些菌株产生并耐受高水平的丁醇。有前景的重组菌株将在发酵研究中使用基于 DNA 阵列的转录、Western 和通量分析进行表征。第二个目标是使用 DNA 阵列和“系统生物学”方法来识别可能参与并赋予溶剂耐受性的其他基因。提出的两个策略构成了目标 2 和 3。第一个策略涉及构建基于质粒的基因组文库（具有不同的复制起点）、转化到亲本生物体中以及鉴定那些赋予所需表型的质粒/基因。该方法建立并扩展了最近提出的方法，并采用了基于 DNA 阵列的选择过程。其次，全基因组 DNA 阵列将用于捕获四种不同溶剂耐受菌株的转录程序的全局图景，以便识别可能与耐受表型相关的常见过表达基因。