Metabolic Engineering of Solvent Tolerance in Anaerobic Bacteria
E. Terry Papoutsakis
Northwestern University

Understanding solvent (and other toxic chemical) tolerance of microorganisms is crucial for the production of chemicals, bioremediation, and whole-cell biocatalysis. It is also very important basic knowledge. Past efforts to produce tolerant strains have relied on selection under applied pressure and chemical mutagenesis, with some good results, but not consistently so.  We desire to examine if Metabolic Engineering (ME) and genomic approaches can be used to construct more tolerant strains for bioprocessing. The accepted dogma is that toxicity is due to the chaotropic effects of solvents on the cell membrane. Impaired membrane fluidity and function inhibit cell metabolism, and result in cell death. We have found that in C. acetobutylicum, several well-defined genetic modifications not related to membrane function impart solvent tolerance (by 40-70%) without strain selection. This suggests that we need to re-examine the accepted dogma. The objective of this research is to identify genes that contribute to solvent tolerance and to use genetic modifications (involving these genes) to generate solvent tolerant strains.  In view of the large number of possible genes that may be involved in determining solvent tolerance, we use DNA microarrays based on the genome sequence of C. acetobutylicum. DNA microarrays were designed and constructed in our laboratory in order to examine the large-scale transcriptional program of the cells in response to various levels of butanol and other solvent challenges. Many genes belonging to several classes (molecular pumps, chaperonins (HSPs), primary metabolism, ATPases, sporulation, transcriptional regulators, carbohydrate metabolism) were identified as changing gene expression under solvent stress. Several of these genes will be explored in ME studies.

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