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

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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.