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

本项目旨在了解和利用决定工业上重要的厌氧梭菌对溶剂耐受性的分子基础。此外，主要研究者（PI）的目标是开发一般的基因组和代谢工程策略，以了解耐化学品的分子基础和开发耐受菌株。PI的假设是，使细菌细胞能够承受高浓度溶剂的分子基础可以用于代谢工程细胞，使它们能够耐受更高浓度的溶剂和相关化学物质。对于这些研究，PI将使用丙酮丁醇梭菌，其基因组已被测序。根据当前资助期的数据，热休克（应激）蛋白（HSP）的过度表达-这是有助于蛋白质折叠和再折叠的分子伴侣-被证明可以提高生产和耐受高丁醇浓度的能力，以及延长细胞代谢。这些特征可能是由于热休克蛋白对细胞机器的稳定作用。该项目的第一个具体目标是使用HSP和代谢基因的伴随过表达来产生并耐受高水平丁醇的菌株。有前途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。第二个目标是使用DNA阵列和“系统生物学”方法来鉴定可能参与并赋予溶剂耐受性的其他基因。这两项拟议战略构成目标2和3。第一种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，并鉴定赋予所需表型的那些质粒/基因。这种方法建立在最近提出的方法的基础上，并扩展了该方法，并采用了基于DNA阵列的选择过程。其次，全基因组DNA阵列将被用来捕捉四种不同的耐溶剂菌株的转录程序的全局图片，以确定通常可能与耐受表型相关的过表达基因。

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.