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.

该项目是要理解和利用分子基础，以决定对溶剂的工业重要厌氧封闭性的耐受性。此外，主要研究人员（PIS）旨在制定一般的基因组和代谢工程策略，以了解对化学物质耐受性和发展耐受菌株的分子基础。 PIS的假设是，使细菌细胞能够承受高溶剂浓度的分子基础可以用于代谢工程细胞，以便它们可以忍受较高浓度的溶剂和相关化学物质。在这些研究中，PIS将采用乙梭菌的基因组进行了测序。基于当前资助期的数据，热震（应激）蛋白（HSP）的过表达（HSPS） - 是分子伴侣，辅助蛋白质折叠和重新折叠 - 可表达增加产生和耐受高丁醇浓度的能力，以及延长细胞代谢的能力。这些特征可能是由于HSP对细胞机械的稳定作用所致。该项目的第一个具体目的是使用HSP和代谢基因的伴随过表达来产生菌株，这些菌株产生和耐受高水平的丁醇。有希望的重组菌株将在使用基于DNA阵列的转录，西方和通量分析的发酵研究中表征。第二个目标是使用DNA阵列和一种“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。提出的两种策略构成目标2和3。第一个策略涉及基于质粒的基因组文库的构建（复制的起源不同），转化为母体生物体以及对这些质粒/基因的识别，这些质粒/基因赋予了理想的表型。这种方法基于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性菌株的转录程序的全局图像，以识别可能与耐受性表型相关的常见过表达的基因。